Heparan sulfate glycosaminoglycan lyase and uses thereof

ABSTRACT

The invention provides recombinant  B. thetaiotaomicron  GAG lyase polypeptides. The invention also provides nucleic acid molecules encoding such polypeptides, recombinant expression vectors containing  B. thetaiotaomicron  GAG lyase nucleic acid molecules, and host cells into which the expression vectors have been introduced. Characterization, diagnostic and therapeutic methods utilizing compositions of the invention are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/436,751filed May 6, 2009, which is a divisional of U.S. application Ser. No.11/948,561 filed Nov. 30, 2007, which is a divisional of U.S.application Ser. No. 11/592,622 filed Nov. 3, 2006, which is acontinuation-in-part application of and claims priority to U.S.application Ser. No. 11/265,908, filed on Nov. 3, 2005.

BACKGROUND OF THE INVENTION

Heparin and heparan sulfate represent a class of glycosaminoglycanscharacterized by a linear polysaccharide of D-glucosamine linked tohexuronic acid (Linhardt, R. J. (1991) Chem. Ind. 2, 45-50; Casu, B.(1985) Adv. Carbohydr. Chem. Biochem. 43, 51-134). Heparin and heparansulfate are complex carbohydrates that play an important functional rolein the extracellular matrix of mammals. These polysaccharides modulateand regulate critical biochemical signaling pathways which impinge onnormal physiological processes such as cell and tissue morphogenesis,cell-cell interactions, and growth and differentiation. Thesepolysaccharides also play a critical role in various pathologiesincluding wound healing, tumor growth and metastasis, certainneurodegenerative disorders and microbial pathogenesis, to name a few.

Much of the current understanding of heparin and heparan sulfatesequence has relied on studies of their biosynthesis (Linhardt, R. J.,Wang, H. M., Loganathan, D., and Bae, J. H. (1992) Biol. Chem. 267,2380-2387; Lindahl, U., Feingold, D., and Roden, L. (1986) TrendsBiochem. Sci. 11, 221-225; Jacobson, I., and Lindahl U. (1980) J. Biol.Chem. 255, 5094-5100; Lindahl, U., and Kjellen, L. (1987) in The Biologyof Extracellular Matrix Proteoglycans (Wight, T. N., and Mecham R., eds)pp. 59-104, Academic Press, New York).

Heparan sulfate, which is chemically related to heparin, is present onthe cell surface and within the extracellular matrix (ECM) of virtuallyevery mammalian cell type. These heparin-like glycosaminoglycans(HLGAGs) are present in this extracellular environment asprotein-polysaccharide conjugates known as proteoglycans. It isincreasingly recognized that HLGAGs play much more than a merestructural role as they interact in a functional manner with numerousproteins of the extracellular matrix, such as laminin, fibronectin,integrins, and collagen. As such, HLGAGs (as part of proteolycans) helpto define the biological properties of the matrix. These HLGAGs alsointeract with an array of cytokine-like growth factors and morphogenspresent within the extracellular matrix by facilitating theirbiochemical interaction with receptors and by protecting them fromproteolytic degradation. For example, heparin potentates the biologicalactivity of aFGF, as reported by Thornton, et al., Science 222, 623-625(1983), possibly by potentiating the affinity of aFGF for its cellsurface receptors, as reported by Schreiber, et al., Proc. Natl. Acad.Sci. USA 82, 6138-6142 (1985). Heparin protects aFGF and bFGF fromdegradation by heat, acid and proteases, as reported by Gospodarowiczand Cheng, J. Cell Physiol. 128, 475-4 84 (1986); Rosengart, et al.,Biochem. Biophys. Res. Commun 152, 432-440 (1988); and Lobb Biochem. 27,2572-2578 (1988). bFGF is stored in the extracellular matrix and can bemobilized in a biologically active form by the hydrolyzing activity ofenzymes such as heparanase as reported by Vlodavsky, et al., Proc. Natl.Acad. Sci. USA 84, 2292-2296 (1987) and Folkman, et al., Am. J. Pathol.130, 393-400 (1988) and Emerson et. al. Proc. Natl. Acad. Sci. USA101(14): 4833-8 (2004).

The binding of FGF to heparan sulfate is a prerequisite for the bindingof FGF to its high affinity receptor on the cell surface, as reported byYayon, et al., Cell 64, 841-848 (1991) and Papraeger, et al., Science252, 1705-1708 (1991). A specific heparan sulfate proteoglycan has beenfound to mediate the binding of bFGF to the cell surface, as describedby Kiefer, et al., Proc. Natl. Acad. Sci. USA 87, 6985-6989 (1990).

Heparin lyases, such as heparinases, are a general class of enzymes thatare capable of specifically cleaving the major glycosidic linkages inheparin and heparan sulfate. Three heparinases have been identified inFlavobacterium heparinum, a GAG-utilizing organism that also producesexoglycuronidases, glycosidases, sulfoesterases, and sulfamidases andother enzymes which further act on the lyase-generated oligosaccharideproducts (Yang, et al. J. Biol. Chem. 260, 1849-1857 (1987); Galliher,et al. Eur. J. Appl. Microbiol. Biotechnol. 15, 252-257 (1982). Theselyases are designated as heparinase I (heparinase, EC 4.2.2.7),heparinase II (heparinase II, no EC number) and heparinase III(heparinase EC 4.2.2.8). The three purified heparinases differ in theircapacity to cleave heparin and heparan sulfate: heparinase I primarilycleaves heparin, heparinase III specifically cleaves heparan sulfate,and heparinase II acts on both heparin and heparan sulfate. SeveralBacteroides species (Saylers, et al. Appl. Environ. Microbiol. 33,319-322 (1977); Nakamura, et al. J. Clin. Microbiol. 26, 1070-1071(1988)) also produce heparin lyases. A heparin lyase has also beenpurified to apparent homogeneity from an unidentified soil bacterium byBohmer, et al. J. Biol. Chem. 265, 13609-13617 (1990).

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery and recombinantexpression of glucosaminoglycan (GAG) lyases from Bacteroidesthetaiotaomicron, hereafter referred to as “B. thetaiotaomicron GAGlyases”, e.g., B. thetaiotaomicron GAG lyase I, B. thetaiotaomicron GAGlyase II B. thetaiotaomicron GAG lyase III, and B. thetaiotaomicron GAGlyase IV, useful, inter alia, in the structure-specific cleavage ofheparin and/or heparan sulfate, and, in some cases, chondroitin sulfateand dermatan sulfate. Thus, the invention includes methods, compositionsand kits with a B. thetaiotaomicron GAG lyase or functional fragmentsthereof and combinations of B. thetaiotaomicron GAG lyases or functionalfragments thereof, for, e.g., characterization or modification ofglycosaminoglycans (GAGs) such as heparin-like glycoaminoglycans(HLGAGs), e.g., heparin and heparan sulfate or, e.g., characterizationor modification of non-heparin/heparan sulfate GAGs, e.g., chondroitinsulfate and dermatan sulfate. For example, the methods, compositions andkits can be used to analyze and monitor heterogeneous populations ofGAGs, e.g., HLGAGs. In other aspects, the methods, compositions and kitscan be used to modify the structure and/or activity of GAGs, e.g.,HLGAGs.

Accordingly, in one aspect, the invention features B. thetaiotaomicronGAG lyase polypeptides, or functional fragments thereof, e.g., B.thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, having the amino acid sequence shown in SEQ ID NOs:2, 4, 6, 8,10, 23, 29, 34, 37 or 39; an amino acid substantially identical to theamino acid sequence shown in SEQ ID NOs:2, 4, 6, 8, 10, 23, 29, 34, 37or 39; or an amino acid encoded by a nucleic acid molecule thathybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising the nucleic acid sequence of SEQ ID NOs:1, 3, 5, 7,9, 22, 28, 33, 36 or 38, wherein the nucleic acid encodes a full lengthB. thetaiotaomicron GAG lyase protein, or functional fragments thereof.

In another aspect, the invention features a composition that includes aB. thetaiotaomicron GAG lyase polypeptide, B. thetaiotaomicron GAG lyasepolypeptides, or functional fragments thereof, e.g., B. thetaiotaomicronGAG lyase polypeptides, B. thetaiotaomicron GAG lyase polypeptides, orfunctional fragments thereof, described herein. In one embodiment, thecomposition further comprises one or more HLGAG degrading enzyme, e.g.,one or more heparinase and/or one or more GAG lyase polypeptide otherthan a B. thetaiotaomicron GAG lyase polypeptides. For example, thecomposition can further include one or more of: an unsaturatedglucuronyl hydrolase (e.g., F. heparinum Δ4,5 glycuronidase; B.thetaiotaomicron Δ4,5 glycuronidase); a glucuronyl hydrolase (e.g.,mammalian α-iduronidase, β-glucuronidase); a sulfohydrolase (e.g., F.heparinum 2-O-sulfatase, 6-O-sulfatase, 3-O-sulfatase: B.thetaiotaomicron 6-O-sulfatase; mucin desulfating enzymes; mammalianN-acetylglucosamine-6-sulfatase; mammalian iduronic acid-2-sulfatase);an N-sulfamidase (e.g., F. heparinum N-sulfamidase; mammalianheparan-N-sulfatase); an arylsulfatase; a hexosaminidase; a glycosylhydrolase (e.g., endo-N-acetyl glucosaminidase); a heparinase (e.g.,Flavobacterum heparinum heparinase I, Flavobacterum heparinum heparinaseII, Flavobacterum heparinum heparinase III, Flavobacterum heparinumheparinase IV aka heparinases from Cytophaga heparina or Pedobacterheparinum), mammalian heparanase, bacteriophage K5 heparan lyase, andfunctional fragments and variants thereof. Such compositions can beused, e.g., to cleave a HLGAG such as heparin and/or heparan sulfate,e.g., to characterize a preparation of HLGAGs such as heparin and/orheparan sulfate.

In another aspect, the invention features a method of specificallycleaving an HLGAG, e.g., heparin or heparan sulfate, that includescontacting an HLGAG with a B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, e.g., a B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, described herein. In one embodiment, the HLGAG is cleaved intodi-, tri-, tetra-, penta-, hexa-, octa-, and/or deca-saccharides and,e.g., the method further includes determining the sequence of the di-,tri-, tetra-, penta-, hexa-, octa-, deca- and/or longer saccharides ofthe HLGAG. In one embodiment, the method further includes contacting theHLGAG with one or more HLGAG degrading enzyme, e.g., a heparinasepolypeptide or a GAG lyase polypeptide other than a B. thetaiotaomicronGAG lyase polypeptide. For example, the HLGAG degrading enzyme can beone or more of: an unsaturated glucuronyl hydrolase (e.g., F. heparinumΔ4,5 glycuronidase; B. thetaiotaomicron Δ4,5 glycuronidase); aglucuronyl hydrolase (e.g., mammalian α-iduronidase, β-glucuronidase); asulfohydrolase (e.g., F. heparinum 2-O-sulfatase, 6-O-sulfatase,3-O-sulfatase: B. thetaiotaomicron 6-O-sulfatase; mucin desulfatingenzymes; mammalian N-acetylglucosamine-6-sulfatase; mammalian iduronicacid-2-sulfatase); a N-sulfamidase (e.g., F. heparinum N-sulfamidase;mammalian heparan-N-sulfatase); an arylsulfatase; a hexosaminidase; aglycosyl hydrolase (e.g., endo-N-acetyl glucosaminidase); a heparinase(e.g., Flavobacterum heparinum heparinase I, Flavobacterum heparinumheparinase II, Flavobacterum heparinum heparinase III, Flavobacterumheparinum heparinase IV aka heparinases from Cytophaga heparina orPedobacter heparinum), mammalian heparanase, bacteriophage K5 heparanlyase, and functional fragments and variants thereof.

In another aspect, the invention features methods for analyzingheterogeneous populations of HLGAGs, e.g., heparin (e.g., UFH, LMWH, andsynthetic heparins), and heparan sulfate, that include contacting thepopulation with a B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, e.g., a B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, described herein. Thus, in some aspects, the invention relatesto methods and products associated with analyzing and monitoringheterogeneous populations of HLGAGs, e.g., to thus defining thestructural signature and activity of heterogeneous populations ofHLGAGs, using a B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, e.g., a B. thetaiotaomicron GAG lyase polypeptide, orfunctional fragment thereof, described herein.

In some embodiments, the method includes determining the structuralsignature of one or more batches of an HLGAG product that has beencontacted with a B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides or functional fragments thereof,e.g., a B. thetaiotaomicron GAG lyase polypeptide, B. thetaiotaomicronGAG lyase polypeptides, or functional fragments thereof, describedherein. In some embodiments, the method further includes selecting abatch as a result of the determination. In some embodiments, the methodfurther includes comparing the results of the determination topreselected values, e.g., a reference standard. The preselected valuecan be, e.g., the presence or absence or a set value (e.g., mole % orarea under the curve) of one or more di-, tri-, tetra-, penta-, hexa-,octa-, and/or deca-saccharide associated with cleavage of the HLGAG witha B. thetaiotaomicron GAG lyase polypeptide, B. thetaiotaomicron GAGlyase polypeptides, or functional fragments thereof, e.g., a B.thetaiotaomicron GAG lyase polypeptide, B. thetaiotaomicron GAG lyasepolypeptides, or functional fragment thereof, described herein.

For any of the methods described herein, a completely or partially B.thetaiotaomicron GAG lyase polypeptide (or polypeptides) digested samplecan be analyzed to determine the structural signature by, e.g., one ormore of mass spectroscopy (e.g., matrix assisted laserdesorption/ionization mass spectroscopy (MALDI-MS)), nuclear magneticresonance (NMR) spectroscopy (e.g., 1D NMR or 2D NMR), gelelectrophoresis, capillary electrophoresis (CE), reverse-phase columnchromatography (e.g., HPLC, e.g., HPLC with a stationary phasedynamically coated with a quanternary ammonium salt), ion-pair HPLC,e.g., strong anion exchange HPLC (SAX-HPLC). The methods describedherein can further include digesting the sample with one or more HLGAGdegrading enzyme, e.g., a heparinase or a heparin lyase polypeptideother than a B. thetaiotaomicron GAG lyase polypeptide. For example, theHLGAG degrading enzyme can be one or more of: an unsaturated glucuronylhydrolase (e.g., F. heparinum Δ4,5 glycuronidase; B. thetaiotaomicronΔ4,5 glycuronidase); a glucuronyl hydrolase (e.g., mammalianα-iduronidase, β-glucuronidase); a sulfohydrolase (e.g., F. heparinum2-O-sulfatase, 6-O-sulfatase, 3-O-sulfatase: B. thetaiotaomicron6-O-sulfatase; mucin desulfating enzymes; mammalianN-acetylglucosamine-6-sulfatase; mammalian iduronic acid-2-sulfatase); aN-sulfamidase (e.g., F. heparinum N-sulfamidase; mammalianheparan-N-sulfatase); an arylsulfatase; a hexosaminidase; a glycosylhydrolase (e.g., endo-N-acetyl glucosaminidase); a heparinase (e.g.,Flavobacterum heparinum heparinase I, Flavobacterum heparinum heparinaseII, Flavobacterum heparinum heparinase III, Flavobacterum heparinumheparinase IV aka heparinases from Cytophaga heparina or Pedobacterheparinum), mammalian heparanase, bacteriophage K5 heparan lyase, andfunctional fragments and variants thereof.

In another aspect, the invention features an HLGAG preparation (e.g., aheparin or heparan sulfate preparation) produced by contacting an HLGAGpreparation with a B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, e.g., a B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, described herein. In one embodiment, the HLGAG preparation(e.g., the heparin or heparan sulfate preparation) has one or more ofreduced anti-Xa activity and anti-IIa activity, e.g., as compared to areference standard, e.g., as compared to a commercially availableheparin or heparan sulfate or as compared to the heparin or heparansulfate preparation prior to contacting with a B. thetaiotaomicron GAGlyase polypeptide. In some embodiments, anti-Xa activity is reducedwhile anti-IIa activity is maintained or increased. In otherembodiments, anti-IIa activity is reduced while anti-Xa activity ismaintained or enhanced. In other embodiments, anti-Xa activity andanti-IIa activity are reduced. Such preparation can be useful, e.g., forapplications where reduced anti-Xa activity and/or anti-IIa activity isdesirable, e.g., such as the use of heparin or heparan sulfate as acarrier for another agent, e.g., a therapeutic agent, prophylactic ordiagnostic agent. Thus, in some embodiments, the HLGAG preparation canfurther include a second agent other than the HLGAG, e.g., thepreparation can further include one or more therapeutic, prophylactic ordiagnostic agents. In another embodiment, the HLGAG preparation (e.g.,the heparin or heparan sulfate preparation) has one or more of increasedanti-Xa activity and anti-IIa activity, e.g., as compared to a referencestandard, e.g., as compared to a commercially available heparin orheparan sulfate or as compared to the heparin or heparan sulfatepreparation prior to contacting with a B. thetaiotaomicron GAG lyasepolypeptide. Such preparation can be useful, e.g., for applications wereincreased anti-Xa activity and/or anti-IIa activity is desirable, e.g.,as an anti-coagulant and/or anti-thrombotic agent.

In another aspect, the invention features a method of neutralizing oneor more activities of an HLGAG (e.g., a heparin or heparan sulfate). Themethod includes contacting the HLGAG with a B. thetaiotaomicron GAGlyase polypeptide, B. thetaiotaomicron GAG lyase polypeptides orfunctional fragments thereof, e.g., a B. thetaiotaomicron GAG lyase Ipolypeptide, B. thetaiotaomicron GAG lyase III polypeptide, a B.thetaiotaomicron GAG lyase IV polypeptide and/or functional fragmentthereof, described herein. When the HLGAG is heparin or heparan sulfate,the activity to be neutralized can be one or more of anti-Xa activityand anti-IIa activity. In some embodiments, anti-Xa activity is reducedwhile anti-IIa activity is maintained or increased. In otherembodiments, anti-IIa activity is reduced while anti-Xa activity ismaintained or enhanced. In other embodiments, anti-Xa activity andanti-IIa activity are reduced. In other embodiments, anti Xa andanti-IIa activities are maintained. The HLGAG can be, e.g., contacted exvivo or in vivo. Thus, in some embodiments, the method can includeadministering the B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides or functional fragments thereof,to a subject in an amount effective to neutralize anti-Xa activityand/or anti-IIa activity in the subject, e.g., a subject that has beenadministered an HLGAG such as heparin or heparan sulfate, e.g., asubject that has been administered heparin or heparan sulfate to inhibitcoagulation and/or thrombosis.

In another aspect, the invention features a method of inhibitingangiogenesis in a subject. The method includes administering to thesubject an effect amount of a B. thetaiotaomicron GAG lyase polypeptide,B. thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, e.g., a B. thetaiotaomicron GAG lyase I polypeptide, a B.thetaiotaomicron GAG lyase II polypeptide, a B. thetaiotaomicron GAGlyase III polypeptide, a B. thetaiotaomicron GAG lyase IV polypeptide,or functional fragments thereof, described herein, to thereby inhibitangiogenesis. In one embodiment, the subject has a disease or disorderassociated with unwanted angiogenesis. Such disorders include, but arenot limited to, arthritis (e.g., rheumatoid arthritis), various eyedisorders (e.g., diabetic retinopathy, neovascular glaucoma,inflammatory disorders, ocular tumors (e.g., retinoblastoma),retrolental fibroplasias, uveitis as well as disorders associated withchoroidal neovascularization and iris neovascularization) and cancer(e.g., tumor growth and metastases).

In another aspect, the invention features a method of inhibitingunwanted cellular proliferation and/or differentiation in a subject. Themethod includes administering to the subject an effect amount of a B.thetaiotaomicron GAG lyase polypeptide, B. thetaiotaomicron GAG lyasepolypeptides or functional fragment thereof, e.g., a B. thetaiotaomicronGAG lyase polypeptide, B. thetaiotaomicron GAG lyase polypeptides, orfunctional fragments thereof, described herein, to thereby inhibitcellular proliferation and/or differentiation. In one embodiment, thesubject has cancer.

In another aspect, the invention features a pharmaceutical compositionthat includes a B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, e.g., a B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, described herein, and a pharmaceutically acceptable carrier. Inone embodiment, the B. thetaiotaomicron GAG lyase polypeptide, B.thetaiotaomicron GAG lyase polypeptides, or functional fragmentsthereof, is present in an amount effective to neutralize one or moreactivity of an HLGAG. Preferably, the HLGAG is heparin or heparansulfate and the B. thetaiotaomicron GAG lyase polypeptide, or functionalfragment thereof, is present in an amount effective to neutralize one ormore of anti-Xa activity and anti-IIa activity. In some embodiments,anti-Xa activity is reduced while anti-IIa activity is maintained orincreased. In other embodiments, anti-IIa activity is reduced whileanti-Xa activity is maintained or enhanced. In other embodiments,anti-Xa activity and anti-IIa activity is reduced. In anotherembodiment, the B. thetaiotaomicron GAG lyase polypeptide, or functionalfragment thereof, is present in an amount effective to inhibitangiogenesis.

In another aspect, the invention features a kit comprising a compositionof B. thetaiotaomicron GAG lyase polypeptide, B. thetaiotaomicron GAGlyase polypeptides, or functional fragments thereof. In one embodiment,the kit further includes one or more HLGAG degrading enzyme, e.g., oneor more heparinase polypeptide and/or one or more GAG lyase polypeptideother than B. thetaiotaomicron GAG lyase polypeptide. For example, thekit can further comprise one or more of: an unsaturated glucuronylhydrolase (e.g., F. heparinum Δ4,5 glycuronidase; B. thetaiotaomicronΔ4,5 glycuronidase); a glucuronyl hydrolase (e.g., mammalianα-iduronidase, β-glucuronidase); a sulfohydrolase (e.g., F. heparinum2-O-sulfatase, 6-O-sulfatase, 3-O-sulfatase: B. thetaiotaomicron6-O-sulfatase; mucin desulfating enzymes; mammalianN-acetylglucosamine-6-sulfatase; mammalian iduronic acid-2-sulfatase); aN-sulfamidase (e.g., F. heparinum N-sulfamidase; mammalianheparan-N-sulfatase); an arylsulfatase; a hexosaminidase; a glycosylhydrolase (e.g., endo-N-acetyl glucosaminidase); a heparinase (e.g.,Flavobacterum heparinum heparinase I, Flavobacterum heparinum heparinaseII, Flavobacterum heparinum heparinase III, Flavobacterum heparinumheparinase IV aka heparinases from Cytophaga heparina or Pedobacterheparinum), mammalian heparanase, bacteriophage K5 heparan lyase, andfunctional fragments and variants thereof. In one embodiment, the B.thetaiotaomicron GAG lyase polypeptide, or functional fragment thereof,and one or more of the other HLGAG degrading enzymes are in the samecomposition. In another embodiment, the B. thetaiotaomicron GAG lyasepolypeptide, or functional fragment thereof, and the other HLGAGdegrading enzyme are in different compositions. In another embodiment,the B. thetaiotaomicron GAG lyase polypeptide, or functional fragmentthereof, is in a pharmaceutical composition with a pharmaceuticallyeffective carrier. The kits can further include an HLGAG, e.g., heparinand/or heparan sulfate. In one embodiment, when the kit includes apharmaceutical composition of a B. thetaiotaomicron GAG lyasepolypeptide, or functional fragment thereof, the HLGAG, e.g., heparinand/or heparan sulfate, is also in a pharmaceutical composition and,e.g., the kit further includes instructional material for neutralizingone or more activity of the HLGAG.

In another aspect, the invention features a nucleic acid molecule whichencodes a B. thetaiotaomicron GAG lyase polypeptides, or functionalfragments thereof. In one embodiment, the isolated nucleic acid moleculeencodes a polypeptide having the amino acid sequence of SEQ ID NOs:2, 4,6, 8, 10, 23, 29, 34, 37 or 39. In other embodiments, the inventionprovides isolated B. thetaiotaomicron GAG lyase nucleic acid moleculeshaving the nucleotide sequence shown in SEQ ID NOs:1, 3, 5, 7, 9, 22,28, 33, 36 or 38. In another embodiment, the invention provides nucleicacid molecules that are substantially identical to (e.g., naturallyoccurring allelic variants) to the nucleotide sequence shown in SEQ IDNOs:1, 5, 28, or 36 and nucleic acid molecules that hybridize understringent hybridization conditions to a nucleic acid molecule comprisingthe nucleotide sequence of SEQ ID NOs:1, 3, 5, 7, 9, 22, 28, 33, 36 or38, wherein the nucleic acid encodes a full length B. thetaiotaomicronGAG lyase protein, or functional fragments thereof.

In a related aspect, the invention further provides nucleic acidconstructs which include a B. thetaiotaomicron GAG lyase nucleic acidmolecule described herein. In certain embodiments, the nucleic acidmolecules of the invention are operatively linked to native orheterogenous regulatory sequences. Also included are vectors and hostcells containing the B. thetaiotaomicron GAG lyase nucleic acidmolecules of the invention, e.g., vectors and host cells suitable forproducing B. thetaiotaomicron GAG lyase nucleic acid molecules andpolypeptides.

In another aspect, the invention features antibodies and antigen-bindingfragments thereof, that react with, or more preferably, specificallybind B. thetaiotaomicron GAG lyase polypeptides.

In another aspect, the invention provides methods of screening forcompounds that modulate the expression or activity of the B.thetaiotaomicron GAG lyase polypeptides or nucleic acids.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a DNA sequence (SEQ ID NO:1) encoding B.thetaiotaomicron GAG lyase I. Initiating methinione codon (ATG) isunderlined and a second, internal methinione codon is doubled unlinedFIG. 1B depicts its predicted amino acid sequence (SEQ ID NO:2) as wellas indicating in bold the N-terminal amino acid residues of two variantsof B. thetaiotaomicron GAG lyase I referred to as the M17 variant (SEQID NO:4) and the Q26 variant (SEQ ID NO:23).

FIG. 2 depicts a BLAST alignment of B. thetaiotaomicron GAG lyase I (SEQID NO:2) with a heparinase I from Flavobacterium heparinum (SEQ IDNO:24) and a consensus sequence (SEQ ID NO:26).

FIG. 3A depicts a DNA sequence (SEQ ID NO:3) encoding the M17 variant ofB. thetaiotaomicron GAG lyase I with the ATG codon for methionine 17(M17) shaded. FIG. 3B depicts its predicted amino acid sequence (SEQ IDNO:4).

FIG. 4A depicts a DNA sequence (SEQ ID NO:22) encoding the Q26 variantof B. thetaiotaomicron GAG lyase I. FIG. 4B depicts its predicted aminoacid sequence (SEQ ID NO:23).

FIG. 5A depicts a DNA sequence (SEQ ID NO:5) encoding B.thetaiotaomicron GAG lyase II. FIG. 5A also depicts portions of thenucleotide sequence encoding B. thetaiotaomicron GAG lyase II that arenot present in two variants of B. thetaiotaomicron GAG lyase II, namelythe “Q23 variant” (SEQ ID NO:7) the deleted portion indicated byunderlining, and the “K169 variant” (SEQ ID NO:9) the deleted portionindicated by shading. FIG. 5B depicts the predicted amino acid sequenceB. thetaiotaomicron GAG lyase II (SEQ ID NO:6) as well as indicating theportions deleted from the amino acid sequence of the Q23 variant (SEQ IDNO:8) and the K169 variant (SEQ ID NO:10).

FIG. 6 depicts a BLAST alignment of B. thetaiotaomicron GAG lyase II(SEQ ID NO:6) with a heparinase III from Flavobacterium heparinum (SEQID NO:25) and a consensus sequence (SEQ ID NO:27).

FIG. 7 is a representation of a MALDI-MS mass spectrum. Panel A depictsthe peaks of untreated ATIII pentasaccharide ARIXTRA®, the structure ofwhich is also shown. Panel B depicts the peaks produced after ARIXTRA®was digested with recombinant B. thetaiotaomicron GAG lyase I. Apentasulfated trisaccharide product, the structure of which is shown,results after digestion.

FIG. 8 depicts a DNA sequence (SEQ ID NO:28) for coding sequence of GAGlyase III gene cloned from Bacteroides thetaiotaomicron. Initiatingmethionine (ATG) and stop (TAA) codons are noted in bold. Codon (CAG)corresponding to glutamine 23 (Q23) is underlined.

FIG. 9 depicts an amino acid sequence (SEQ ID NO:29) of GAG lyase IIIcloned from Bacteroides thetaiotaomicron. Predicted export signalsequence is underlined. Glutamine 23 (Q23) delimiting beginning ofamino-terminal variant is shaded in gray.

FIG. 10 depicts alignment of amino acid sequence of Bacteroidesthetaiotaomicron GAG lyase III (SEQ ID NO:29)—listed third from thetop—with related heparin/heparan sulfate glycosaminoglycan lyases.Identical residues are shown in dark gray; similar residues are shown inlight gray. BT represents Bacteroides thetaiotaomicron; FH representsFlavobacterium heparinum.

FIG. 11 depicts enzyme activities of GAG lyase III isolated fromBacteroides thetaiotaomicron (BT GAG Lyase), heparinase II isolated fromFlavobacterium heparinum (FH Heparinase II) and heparinase III isolatedfrom Flavobacterium heparinum (FH Heparinase III). The enzyme activityrepresents substrate specificity of the three tested enzymes. The four“heparin-like” substrates tested were: porcine intestinal heparin, twodifferent heparan sulfates (designated “HI” and “HO,” each one varyingin the degree of sulfation), and low molecular weight pharmaceuticalheparin, enoxaparin. Enzyme activity shown depicts total cleavageactivity toward these substrates in an exhaustive digestion, as assessedby absorbance at 232 nm.

FIG. 12 depicts cleavage activities of GAG lyase III isolated fromBacteroides thetaiotaomicron (BT GAG Lyase) and heparinase II isolatedfrom Flavobacterium heparinum (FH Hep II). The cleavage activityrepresents substrate specificity of the tested enzymes. The actualcleavage products are fractionated by capillary electrophoresis andmonitored by absorbance at 232 nm (Y axis). Solid line depicts BT GAGLyase and dotted line depicts FH Hep II.

FIG. 13 depicts a DNA sequence (SEQ ID NO:36) encoding B.thetaiotaomicron GAG lyase IV. Initiating methinione codon (ATG) andstop codon are in bold and a second, codon (GAC) corresponding toaspartate 20 is unlined.

FIG. 14 depicts its predicted amino acid sequence (SEQ ID NO:37) withthe signal sequence underlined. In addition, the N-terminal amino acidresidues of the variant of B. thetaiotaomicron GAG lyase IV referred toas the D20 variant (SEQ ID NO:38) is shaded.

FIG. 15 depicts alignment of nucleic acid sequence of Bacteroidesthetaiotaomicron GAG lyase IV (SEQ ID NO:36)—listed third from thetop—with related heparin/heparan sulfate lyases. Identical residues areshown in dark gray; similar residues are shown in light gray. BTrepresents Bacteroides thetaiotaomicron; FH represents Flavobacteriumheparinum.

FIG. 16 depicts the substrate specificity of Bacteroidesthetaiotaomicron GAG lyase IV against three different substrates, namelyunfractionated heparin (Heparin) and two fractions of heparin sulfate(HS) referred to as “HO-HS” and “HI-HS”.

DETAILED DESCRIPTION Overview

This disclosure describes recombinant expression of active B.thetaiotaomicron GAG lyases from B. thetaiotaomicron, that are useful,inter alia, in the modification and characterization of GAGs such asheparin and/or heparan sulfate glycosaminoglycans and derivativesthereof.

For example, the B. thetaiotaomicron GAG lyases described herein can bea complementary tool to existing chemo-enzymatic methods for cleavingGAGs such as heparin and heparan sulfate polysaccharides (and, in somecases, other GAGs such as chondroitin sulfate and dermatan sulfate) in astructure-specific fashion. Structure specific cleavage of a GAG, e.g.,heparin and/or heparan sulfate, can be used, e.g., to determine thestructure of GAGs in a heterogenous GAG preparation. In addition,cleavage can be used, e.g., to produce lower molecular weightoligosaccharides from the GAG. Thus, the B. thetaiotaomicron GAG lyasescan be used to generate, e.g., heparin- and heparan sulfate-derivedoligosaccharides. Such heparin- and heparan sulfate-derivedoligosaccharides may have diagnostic, prophylactic and therapeuticpotential.

In addition, the B. thetaiotaomicron GAG lyases described herein mayalso have prophylactic and therapeutic potential, e.g., in disordersassociated with angiogenesis.

The B. thetaiotaomicron GAG lyases further can be used in vitro, ex vivoand/or in vivo to neutralize an anti-coagulant and/or anti-thromboticactivity of heparin and/or heparan sulfate.

The B. thetaiotaomicron GAG lyase I sequence (FIG. 1; SEQ ID NO:1),which is approximately 1251 nucleotides long including potentiallyuntranslated regions, contains a predicted methionine-initiated codingsequence of about 1179 nucleotides, including the termination codon(nucleotides indicated as coding of SEQ ID NO:1 in FIG. 1). The putativecoding sequence encodes a 392 amino acid protein (SEQ ID NO:2).

A variant in which the amino terminus begins at the methinione atresidue 17 (M17) can also be used to produce recombinant protein. Theamino acid sequence and nucleotide sequence encoding the M17 variant ofB. thetaiotaomicron GAG lyase I are depicted in FIG. 3B (SEQ ID NO:4)and 3A (SEQ ID NO:3), respectively. In addition, a 6× histidine fusionprotein has been generated to facilitate purification. Inclusion ofdifferent purification tags such as GST, MBP, Trx, DsbC, NusA or biotincan also be used to obtain this enzyme.

Another variant in which the amino terminus begins at the glutamine atresidue Q26 can also be used to produce recombinant protein. The aminoacid sequence and nucleotide sequence encoding the Q26 variant of B.thetaiotaomicron GAG lyase I are depicted in FIG. 4B (SEQ ID NO:23) and4A (SEQ ID NO:22), respectively. In addition, a 6× histidine fusionprotein has been generated to facilitate purification. Inclusion ofdifferent purification tags such as GST, MBP, Trx, DsbC, NusA or biotincan also be used to obtain this enzyme.

The B. thetaiotaomicron GAG lyase I protein shares structuralcharacteristics with heparinase I obtained from Flavobacteriumheparinum, at least at the primary amino acid sequence level (FIG. 2).

The B. thetaiotaomicron GAG lyase II gene sequence (FIG. 5; SEQ IDNO:5), which is approximately 2001 nucleotides long inclusive of thetermination codon (nucleotides indicated as coding of SEQ ID NO:5 inFIG. 5A). The coding sequence encodes a 666 amino acid protein (SEQ IDNO:5B).

A variant in which the amino terminus begins at the glutamine at residue23 (Q23) can also be used to produce recombinant protein. The amino acidsequence and nucleotide sequence encoding the Q23 variant of B.thetaiotaomicron GAG lyase II are depicted in FIG. 5B (SEQ ID NO:8) and5A (SEQ ID NO:7), respectively. In addition, a 6× histidine fusionprotein has been generated to facilitate purification. Inclusion ofdifferent purification tags such as GST, MBP, Trx, DsbC, NusA or biotincan also be used to obtain this enzyme.

Another variant in which is a deletion beginning at the lysine atresidue 169 (K169) and ending at the glutamic acid at residue 186 canalso be used to produce recombinant protein. The amino acid sequence andnucleotide sequence encoding the K169 variant of B. thetaiotaomicron GAGlyase II are depicted in FIG. 5B (SEQ ID NO:10) and 5A (SEQ ID NO:9),respectively. The B. thetaiotaomicron GAG lyase II protein shares anumber of structural characteristics with heparinase III obtained fromFlavobacterium heparinum, at least at the level of their respectiveprimary amino acid sequences.

The B. thetaiotaomicron GAG lyase III sequence (FIG. 8; SEQ ID NO:28),which is approximately 2690 nucleotides long (including untranslatedsequence) and contains a predicted methionine-initiated coding sequenceof about 2622 nucleotides, including the termination codon. Theinitiation and the termination codons are depicted in bold in FIG. 8.The coding sequence encodes an 873 amino acid protein (SEQ ID NO:29 inFIG. 9). A variant in which the amino terminus begins at the glutamineresidue 23 (Q23) can also be used to produce recombinant protein. Thenucleotide sequence of the variant is depicted in SEQ ID NO:33, whilethe amino acid sequence of the variant is shown in SEQ ID NO:34.Glutamine 23 residue is underlined in FIG. 9 and shaded in gray in FIG.10. In addition, a 6× histidine fusion protein has been generated tofacilitate purification. Inclusion of different purification tags suchas GST, MBP, Trx, DsbC, NusA and biotin can also be used to obtain thisenzyme.

The B. thetaiotaomicron GAG lyase III protein contains some structuralcharacteristics and substrate specificity in common with heparinase IIobtained from Flavobacterium heparinum. The B. thetaiotaomicron GAGlyase III protein has substrate specificity for both heparin and heparansulfate. In addition, it is capable of cleaving chondroitin sulfate anddermatan sulfate.

The B. thetaiotaomicron GAG lyase IV sequence (FIG. 13; SEQ ID NO:36),which is approximately 2109 nucleotides long. The initiation and thetermination codons are in bold in FIG. 15. The coding sequence encodes a702 amino acid protein (SEQ ID NO:37 in FIG. 14).

A variant in which the amino terminus begins at the aspartate at aminoacid residue 20 (D20) can also be used to produce recombinant protein.The nucleotide sequence of the variant is depicted in SEQ ID NO:38,while the amino acid sequence of the variant is shown in SEQ ID NO:39.The codon for aspartate 20 residue is underlined in FIG. 13 andaspartate 20 is shaded in gray in FIG. 14. In addition, a 6× histidinefusion protein has been generated to facilitate purification. Inclusionof different purification tags such as GST, MBP, Trx, DsbC, NusA orbiotin can also be used to obtain this enzyme.

The B. thetaiotaomicron GAG lyase IV protein contains limited sequencesimilarity with other GAG lyases obtained from B. thetaiotaomicron orheparinases obtained from Flavobacterium heparinum. In addition, B.thetaiotaomicron GAG lyase IV protein has substrate specificity thatvaries from other GAG lyases obtained from B. thetaiotaomicron orheparinases obtained from Flavobacterium heparinum. For example, it cancleave di- or tetrasaccharides typically underrepresented in mostnaturally occurring heparin and/or heparan sulfates reported in thescientific literature.

As the B. thetaiotaomicron GAG lyase polypeptides of the invention maymodulate heparin- and/or heparan sulfate-mediated activities, they maybe useful in various prophylactic and therapeutic applications as wellas for developing novel prophylactic and diagnostic agents for heparin-or heparan sulfate-mediated or related disorders.

As used herein, a “GAG lyase activity”, “biological activity of GAGlyase” or “functional activity of GAG lyase”, refers to an activityexerted by a B. thetaiotaomicron GAG lyase protein, polypeptide ornucleic acid molecule in a physiological milieu. For example, a GAGlyase activity can be an activity exerted by B. thetaiotaomicron GAGlyase on e.g., on a GAG lyase substrate, e.g., glycosidic linkages inheparin or heparan sulfate. A GAG lyase activity can be determined invivo or in vitro.

The B. thetaiotaomicron GAG lyase molecules of the present invention arepredicted to have similar biological activities to various heparinasesobtained from Flavobacterium heparinum. For example, the B.thetaiotaomicron GAG lyase proteins of the present invention can haveone or more of the following activities: (1) binds a heparin and/or aheparan sulfate; (2) cleaves one or more glycosidic linkages of aheparin and/or a heparan sulfate, e.g., in such a manner as to generateat the site of cleavage a uronic acid possessing an unsaturated bondbetween positions C4 and C5 (i.e., Δ4,5); (3) modulates, e.g., increasesor reduces, anti-Xa activity and/or anti-IIa activity of a heparinand/or a heparan sulfate; and (4) reduces or eliminates angiogenesis.

In some aspects, the B. thetaiotaomicron GAG lyase I has biologicalactivity similar to, but not identical with, heparinase I obtained fromFlavobacterium heparinum. For example, the B. thetaiotaomicron GAG lyaseI can have one or more of the following activities: (1) binds a heparinand/or heparan sulfate; (2) cleaves one or more glycosidic linkages ofheparin and/or heparan sulfate, e.g., cleaves one or more glycosidiclinkages involving sulfated uronic acids, e.g., 2-O uronic acids;cleaves one or more glycosidic linkages involving sulfated hexosamines,e.g., 6-O-sulfates and/or N-sulfamides; (3) reduces anti-Xa activityand/or anti-IIa activity of a heparin and/or a heparan sulfate, e.g., ascompared to a reference standard, e.g., the anti-Xa activity and/oranti-IIa activity of a commercially available heparin or heparan sulfateor of the heparin or heparan sulfate prior to cleavage. In someembodiments, anti-Xa activity is reduced while anti-IIa activity ismaintained. In other embodiments, anti-Xa activity and anti-IIa activityare reduced.

In some aspects, the B. thetaiotaomicron GAG lyase III has biologicalactivity similar to, but not identical with, heparinase II obtained fromFlavobacterium heparinum. For example, the B. thetaiotaomicron GAG lyaseIII can have one or more of the following activities: (1) binds aheparin, heparan sulfate, chondroitin sulfate and/or dermatin sulfate;(2) cleaves one or more glycosidic linkages of heparin and/or heparansulfate, e.g., cleaves one or more glycosidic linkages of between asulfated hexosamine (e.g., N-sulfated and/or 6-O sulfated) or anunsulfated, but acetylated hexosamine (e.g., HNAc) and a sulfated uronicacid, e.g., a 2-O sulfated uronic acid, or an unsulfated uronic acid;(3) decreases anti-Xa activity and/or anti-IIa activity of a heparinand/or a heparan sulfate, e.g., as compared to a reference standard,e.g., the anti-Xa activity and/or anti-IIa activity of a commerciallyavailable heparin or heparan sulfate or of the heparin or heparansulfate prior to cleavage. In some embodiments, anti-Xa activity isreduced while anti-IIa activity is possibly maintained. In otherembodiments, anti-Xa activity and anti-IIa activity are both reduced.

Thus, the B. thetaiotaomicron GAG lyase molecules, e.g., the B.thetaiotaomicron GAG lyase I and/or the B. thetaiotaomicron GAG lyaseIII molecules, can act as novel therapeutic agents for controllingheparin-associated disorders. Examples of such disorders includeheparin-induced anticoagulation and/or angiogenesis. For example, the B.thetaiotaomicron GAG lyase molecules, e.g., the B. thetaiotaomicron GAGlyase I and/or the B. thetaiotaomicron GAG lyase III molecules, can beused to reduce or eliminate (e.g., neutralize) one or moreanticoagulation properties of a heparin and/or a heparan sulfate, e.g.,during or after surgery. In other embodiments, the B. thetaiotaomicronGAG lyase molecules, e.g., B. thetaiotaomicron GAG lyase I and/or the B.thetaiotaomicron GAG lyase III, can be used to deheparinize blood, e.g.,in a bioreactor, e.g., a bioreactor used in heart-lung and/or kidneydialysis.

In some aspects, the B. thetaiotaomicron GAG lyase II has biologicalactivity similar to, but not identical with, heparinase III obtainedfrom Flavobacterium heparinum. For example, the B. thetaiotaomicron GAGlyase II can have one or more of the following activities: (1) binds aheparin and/or heparan sulfate; (2) cleaves one or more glycosidiclinkages of heparin and/or heparan sulfate, e.g., cleaves one or moreglycosidic linkages of sulfated and undersulfated uronic acids; (3)increases anti-Xa activity and/or anti-IIa activity of a heparin and/ora heparan sulfate, e.g., as compared to a reference standard, e.g., theanti-Xa activity and/or anti-IIa activity of a commercially availableheparin or heparan sulfate or of the heparin or heparan sulfate prior tocleavage. In some embodiments, anti-Xa activity is maintained orpossibly increased while anti-IIa activity is reduced. In otherembodiments, anti-IIa activity is increased while anti-Xa activity ismaintained.

In some aspects, the B. thetaiotaomicron GAG lyase IV has biologicalactivity, e.g., substrate specificity, distinct from other known GAGlyases obtained from B. thetaiotaomicron and heparinases obtained fromFlavobacterium heparinum. For example, the B. thetaiotaomicron GAG lyaseIV can have one or more of the following activities: (1) binds a heparinand/or heparan sulfate; (2) cleaves one or more glycosidic linkages ofheparin and/or heparan sulfate, e.g., cleaves one or more glycosidiclinkages of low to medium sulfate density, especially linkages involvinga 2-O-sulfated uronic acid and adjoining acetylated glucosamine (notcommonly found in most naturally occurring preparations of heparinand/or heparan sulfate); (3) increases anti-Xa activity and/or anti-IIaactivity of a heparin and/or a heparan sulfate, e.g., as compared to areference standard, e.g., the anti-Xa activity and/or anti-IIa activityof a commercially available heparin or heparan sulfate or of the heparinor heparan sulfate prior to cleavage. In some embodiments, anti-Xaactivity is increased while anti-IIa activity is maintained or reduced.In other embodiments, anti-IIa activity is increased while anti-Xaactivity is maintained.

Thus, such B. thetaiotaomicron GAG lyase molecules, e.g., B.thetaiotaomicron GAG lyase II and B. thetaiotaomicron GAG lyase IVmolecules, can be used to prepare a heparin and/or heparan sulfatepreparation useful for treatment of coagulation and/or thrombosis.Examples of such disorders include dissolving or inhibiting formation ofthromboses, treatment and prevention of conditions resulting frominfarction of cardiac and central nervous system vessels,atherosclerosis, thrombosis, myocardial infarction, arrythmias, atrialfibrillation, angina, unstable angina, refractory angina, congestiveheart failure, disseminated intravascular coagulation, percutaneouscoronary intervention (PCI), coronary artery bypass graft surgery(CABG), reocclusion or restenosis of reperfused coronary arteries,rheumatic fever, stroke, transient ischemic attacks, thrombotic stroke,embolic stroke, deep venous thrombosis, pulmonary embolism, migraine,allergy, asthma, emphysema, adult respiratory stress syndrome (ARDS),cystic fibrosis, neovascularization of the ocular space, osteoporosis,psoriasis, arthritis (rheumatoid or osteogenic), Alzheimer's disease,bone fractures, major surgery/trauma, burns, surgical procedures,transplantation such as bone marrow transplantation, hip replacement,knee replacement, sepsis, septic shock, pregnancy, hereditary disorderssuch as hemophilias.

In other embodiments, the B. thetaiotaomicron GAG lyase molecules, e.g.,B. thetaiotaomicron GAG lyase II and/or B. thetaiotaomicron GAG lyase IVmolecules, can be used to treat or prevent cellular proliferative ordifferentiative disorders, e.g., by preventing or inhibitingangiogenesis of cells exhibiting or otherwise associated with unwantedproliferation and/or differentiation. Examples of cellular proliferativeand/or differentiative disorders include diabetes; arthritis, e.g.,rheumatoid arthritis; ocular disorders, e.g., ocular neovascularization,diabetic retinopathy, neovascular glaucoma, retrolental fibroplasia,uveitis, eye disease associated with choroidal neovascularization, eyedisorders associated with iris neovascularization; cancer, e.g.,carcinoma, sarcoma, metastatic disorders or hematopoietic neoplasticdisorders, e.g., leukemias.

In other embodiments, the B. thetaiotaomicron GAG lyase molecules, e.g.,B. thetaiotaomicron GAG lyase III molecules, can be used to prepare achondroitin sulfate and/or dermatan sulfate preparation.

The B. thetaiotaomicron GAG lyase proteins, fragments thereof, andderivatives and other variants of the sequence in SEQ ID NO:2, SEQ IDNO:6, SEQ ID NO: 29 or SEQ ID NO:37 thereof are collectively referred toas “polypeptides or proteins of the invention” or “B. thetaiotaomicronGAG lyase polypeptides or proteins”. Nucleic acid molecules encodingsuch polypeptides or proteins are collectively referred to as “nucleicacids of the invention” or “B. thetaiotaomicron GAG lyase nucleicacids.” “B. thetaiotaomicron GAG lyase molecules” refer to B.thetaiotaomicron GAG lyase nucleic acids, polypeptides, and antibodies.

As used herein, the term “nucleic acid molecule” includes DNA molecules(e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogsof the DNA or RNA. A DNA or RNA analog can be synthesized fromnucleotide analogs. A DNA molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “isolated nucleic acid molecule” or “purified nucleic acidmolecule” includes nucleic acid molecules that are separated from othernucleic acid molecules present in the natural source of the nucleicacid. For example, with regards to genomic DNA, the term “isolated”includes nucleic acid molecules which are separated from the chromosomewith which the genomic DNA is naturally associated. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatednucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of 5′and/or 3′ nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing. Stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1%SDS at 65° C. Hybridization conditions are known to those skilled in theart and can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methodsare described in that reference and either can be used. Additionalexamples of hybridization conditions are as follows: 1) low stringency,hybridization in 6×SSC at about 45° C., followed by one or more washesin 0.2×SSC, 0.1% SDS at 55° C.; 2) medium stringency, hybridization in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; and preferably, 3) high stringency, hybridization in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 65° C. Particularly preferred stringency conditions (and theconditions that should be used if the practitioner is uncertain aboutwhat conditions should be applied to determine if a molecule is within ahybridization limitation of the invention) are 0.5M sodium phosphate, 7%SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65°C. Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1, 5,28 or 36 corresponds to a naturally-occurring nucleic acid molecule.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature. For example a naturally occurring nucleic acid molecule canencode a natural protein.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include at least an open reading frame encoding aB. thetaiotaomicron GAG lyase protein. The gene can optionally furtherinclude non-coding sequences, e.g., regulatory sequences and introns.

An “isolated” or “purified” polypeptide or protein is substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.“Substantially free” means that a preparation of B. thetaiotaomicron GAGlyase protein is at least 10% pure. In a preferred embodiment, thepreparation of B. thetaiotaomicron GAG lyase protein has less than about30%, 20%, 10% and more preferably 5% (by dry weight), of non-B.thetaiotaomicron GAG lyase protein (also referred to herein as a“contaminating protein”), or of chemical precursors or non-B.thetaiotaomicron GAG lyase chemicals. When the B. thetaiotaomicron GAGlyase protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation. The invention includes isolated orpurified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams indry weight.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of B. thetaiotaomicron GAG lyase withoutabolishing or substantially altering a GAG lyase activity. Preferably,the alteration does not substantially alter the GAG lyase activity,e.g., the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type.An “essential” amino acid residue is a residue that, when altered fromthe wild-type sequence of B. thetaiotaomicron GAG lyase, results inabolishing a GAG lyase activity such that less than 20% of the wild-typeactivity is present. For example, conserved amino acid residues in B.thetaiotaomicron GAG lyase are predicted to be particularly unamenableto alteration.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a B. thetaiotaomicron GAGlyase protein is preferably replaced with another amino acid residuefrom the same side chain family. Alternatively, in another embodiment,mutations can be introduced randomly along all or part of a B.thetaiotaomicron GAG lyase coding sequence, such as by saturationmutagenesis, and the resultant mutants can be screened for GAG lyasebiological activity to identify mutants that retain activity. Followingmutagenesis of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:28, SEQ ID NO:36, theencoded protein can be expressed recombinantly and the activity of theprotein can be determined.

As used herein, a “biologically active portion” of a B. thetaiotaomicronGAG lyase protein includes a fragment of a B. thetaiotaomicron GAG lyaseprotein which participates in an interaction, e.g., an inter-molecularinteraction. An inter-molecular interaction can be a binding interactionor an enzymatic interaction (e.g., the interaction can be transient anda covalent bond is formed or broken). An inter-molecular interaction canbe between a GAG lyase B. thetaiotaomicron molecule and a non-B.thetaiotaomicron GAG lyase molecule, e.g., heparin, heparan sulfate, andfragments thereof. Biologically active portions of a B. thetaiotaomicronGAG lyase protein include peptides comprising amino acid sequencessufficiently homologous to or derived from the amino acid sequence ofthe B. thetaiotaomicron GAG lyase protein, e.g., the amino acid sequenceshown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:29 and SEQ ID NO:37, whichinclude less amino acids than the full length B. thetaiotaomicron GAGlyase proteins, and exhibit at least one activity of a GAG lyaseprotein. Typically, biologically active portions comprise a domain ormotif with at least one activity of the GAG lyase protein, e.g.,depolymerization of heparin, heparan sulfate, and fragments thereof(e.g., in a site specific manor); cleavage of a glycosidic linkage ofheparin, heparan sulfate, and fragments thereof; reduce or eliminate ananticoagulant activity, e.g., an anticoagulant activity of heparin,heparan sulfate, and fragments thereof. A biologically active portion ofa B. thetaiotaomicron GAG lyase protein can be a polypeptide which is,for example, 10, 25, 50, 100, 200, 300, 400, 500 or more amino acids inlength.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap needed to beintroduced for optimal alignment of the two sequences. For the purposesof determining if a molecule is within a sequence identity or a homologylimitation herein, percent identity is determined by the mathematicalalgorithm of Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) asimplemented in the GAP program of the GCG software package (available athttp://www.gcg.com) with the following parameters: a Blossum 62 scoringmatrix with a gap penalty of 12, a gap extend penalty of 4, and aframeshift gap penalty of 5.

In a preferred embodiment, the length of a reference sequence alignedfor comparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, 60%, and even more preferably at least 70%,80%, 90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”).

For the purposes of analyzing a biological sequence with reference to B.thetaiotaomicron GAG lyase molecules, the following alignment procedurescan be used in addition to the aforementioned Needleman and Wunschalgorithm. The percent identity between two amino acid or nucleotidesequences can be determined using the algorithm of E. Meyers and W.Miller ((1989) CABIOS, 4:11-17) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to B.thetaiotaomicron GAG lyase nucleic acid molecules of the invention.BLAST protein searches can be performed with the XBLAST program,score=50, wordlength=3 to obtain amino acid sequences homologous to B.thetaiotaomicron GAG lyase protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov.

Particular B. thetaiotaomicron GAG lyase polypeptides of the presentinvention have an amino acid sequence sufficiently identical to theamino acid sequence of SEQ ID Nos:2, 4, 6, 8, 10, 23, 29, 34, 37 or 39.In the context of an amino acid sequence, the term “sufficientlyidentical” or “substantially identical” is used herein to refer to afirst amino acid that contains a sufficient or minimum number of aminoacid residues that are i) identical to or ii) conservative substitutionsof to aligned amino acid residues in a second amino acid sequence suchthat the first and second amino acid sequences have a common structuralfold and/or a common functional activity. For example, amino acidsequences that contain a common structural domain having at least about60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are termedsufficiently or substantially identical. In the context of nucleotidesequence, the term “sufficiently identical” or “substantially identical”is used herein to refer to a first nucleic acid sequence that contains asufficient or minimum number of nucleotides that are identical toaligned nucleotides in a second nucleic acid sequence such that thefirst and second nucleotide sequences have a common functional activityor encode a common structural polypeptide fold or a common functionalpolypeptide activity.

The methods taught herein are sometimes described with reference toheparin-like glycoaminoglycans (HLGAGs) but the properties taught hereincan be extended to other polysaccharides. As used herein the terms“HLGAG” and “glycosaminoglycans” (GAGs) are used interchangeably torefer to a family of molecules having heparin like structures andproperties, generally referred to herein as “heparin”. These moleculesinclude but are not limited to low molecular weight heparin (LMWH),unfractionated heparin, biotechnologically prepared heparin, chemicallymodified heparin, synthetic heparin such as pentasaccharides (e.g.,ARIXTRA™), heparin mimetics and heparan sulfate. The term“biotechnological heparin” encompasses heparin that is prepared fromnatural sources of polysaccharides which have been chemically modifiedand is described in Razi et al., Bioche. J. 1995 Jul. 15; 309 (Pt 2):465-72. Chemically modified heparin is described in Yates et al.,Carbohydrate Res (1996) Nov. 20; 294:15-27, and is known to those ofskill in the art. Synthetic heparin is well known to those of skill inthe art and is described in Petitou, M. et al., Bioorg Med Chem. Lett.(1999) Apr. 19; 9(8):1161-6 and Vlodavsky et al., Int. J. Cancer, 1999,83:424-431. An example of a synthetic heparin is fondaparinux.Fondaparinux (ARIXTRA™) is a 5 unit synthetic glycoaminoglycancorresponding to the AT-III binding site. Heparan Sulfate refers to aglycoaminoglycan containing a disaccharide repeat unit similar toheparin, but which has more N-acetyl groups and fewer N- and O-sulfategroups. Heparin mimetics are monosaccharides (e.g., sucralfate),oligosaccharides, or polysaccharides having at least one biologicalactivity of heparin (i.e., anticoagulation, inhibition of cancer,treatment of lung disorders, etc.). Preferably these molecules arehighly sulfated. Heparin mimetics may be naturally occurring, syntheticor chemically modified. (Barchi, J. J., Curr. Pharm. Des., 2000, Mar.,6(4):485-501). The term “HLGAG” also encompasses functional variants ofthe above-described HLGAG molecules. These functional variants have asimilar structure but include slight modifications to the structurewhich allow the molecule to retain most of its biological activity orhave increased biological activity.

“LMWH” as used herein refers to a preparation of sulfatedglycosaminoglycans (GAGs) having an average molecular weight of lessthan 8000 Da, with about at least 60% of the oligosaccharide chains of aLMWH preparation having a molecular weight of less than 8000 Da. SeveralLMWH preparations are commercially available, but, LMWHs can also beprepared from heparin, using e.g., HLGAG degrading enzymes. HLGAGdegrading enzymes include but are not limited to heparinase-I,heparinase-II, heparinase-III, heparinase IV, heparanase,D-glucuronidase and L-iduronidase. The three heparinases fromFlavobacterium heparinum are enzymatic tools that have been used for thegeneration of LMWH (5,000-8,000 Da) and ultra-low molecular weightheparin (3,000 Da). In addition, LMWHs can be prepared using, e.g., theB. thetaiotaomicron GAG lyase polypeptides described herein.Commercially available LMWH include, but are not limited to, enoxaparin(brand name Lovenox; Aventis Pharmaceuticals), dalteparin (Fragmin,Pharmacia and Upjohn), certoparin (Sandobarin, Novartis), ardeparin(Normiflo, Wyeth Lederle), nadroparin (Fraxiparine, Sanofi-Winthrop),parnaparin (Fluxum, Wassermann), reviparin (Clivarin, Knoll AG), andtinzaparin (Innohep, Leo Laboratories, Logiparin, Novo Nordisk). Somepreferred forms of LMWH include enoxaparin (Lovenox) and dalteparin(Fragmin). The term “Arixtra” as used herein refers to a compositionwhich includes a synthetic pentasaccharide of methylO-2-deoxy-6-O-sulfo-2-(sulfoamino)-I-D-glucopyranosyl-(1→4)-O-θ-D-glucopyranosyl-(1→4)-O-2-deoxy-3,6-di-O-sulfo-2-(sulfoamino)-I-D-glucopyranosyl-(1→4)-O-2-O-sulfo-I-L-idopyranuronosyl-(1→4)-2-deoxy-6-O-sulfo-2-(sulfoamino)-I-D-glucopyranoside,decasodium salt and derivatives thereof. A “synthetic heparin” or“synthetic HLGAG” as used herein refers to HLGAGs are synthesizedcompounds and are not derived by fragmentation of heparin. Methods ofpreparing synthetic heparins are provided, for example, in Petitou etal. (1999) Nature 398:417, the contents of which is incorporated hereinby reference.

“Misexpression or aberrant expression”, as used herein, refers to anon-wildtype pattern of gene expression at the RNA or protein level. Itincludes: expression at non-wild type levels, i.e., over- orunder-expression; a pattern of expression that differs from wild type interms of the time or stage at which the gene is expressed, e.g.,increased or decreased expression (as compared with wild type) at apredetermined developmental period or stage; a pattern of expressionthat differs from wild type in terms of altered expression (as comparedwith wild type) in a predetermined cell type or tissue type; a patternof expression that differs from wild type in terms of the splicing size,translated amino acid sequence, post-transitional modification, orbiological activity of the expressed polypeptide; a pattern ofexpression that differs from wild type in terms of the effect of anenvironmental stimulus or extracellular stimulus on expression of thegene, e.g., a pattern of increased or decreased expression (as comparedwith wild type) in the presence of an increase or decrease in thestrength of the stimulus.

“Subject”, as used herein, refers to a mammal organism. In a preferredembodiment, the subject is a human. In another embodiment, the subjectis an experimental animal or animal suitable as a disease model.Non-limiting examples of such subjects include mice, rats, and rabbits.The subject can also be a non-human animal, e.g., a horse, cow, goat, orother domestic animal.

Various aspects of the invention are described in further detail below.

Isolated Nucleic Acid Molecules

In one aspect, the invention provides, an isolated or purified, nucleicacid molecule that encodes a B. thetaiotaomicron GAG lyase polypeptidedescribed herein, e.g., a full length B. thetaiotaomicron GAG lyaseprotein or a fragment thereof, e.g., a biologically active portion of B.thetaiotaomicron GAG lyase protein. Also included is a nucleic acidfragment suitable for use as a hybridization probe, which can be used,e.g., to identify a nucleic acid molecule encoding a polypeptide of theinvention, B. thetaiotaomicron GAG lyase mRNA, and fragments suitablefor use as primers, e.g., PCR primers for the amplification or mutationof nucleic acid molecules.

In one embodiment, an isolated nucleic acid molecule of the inventionincludes the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:5, SEQID NO:28 or SEQ ID NO:37 or a portion of any of these nucleotidesequences. In one embodiment, the nucleic acid molecule includessequences encoding the B. thetaiotaomicron GAG lyase protein (i.e., “thecoding region” of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:28 or SEQ IDNO:36), as well as 5′ untranslated sequences. Alternatively, the nucleicacid molecule can include no flanking sequences which normally accompanythe subject sequence. In another embodiment, an isolated nucleic acidmolecule of the invention includes a nucleic acid molecule which is acomplement of the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:5,SEQ ID NO:28 or SEQ ID NO:36, or a portion of any of these nucleotidesequences. In other embodiments, the nucleic acid molecule of theinvention is sufficiently complementary to the nucleotide sequence shownin SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:28 or SEQ ID NO:36, such that itcan hybridize to the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:5, SEQ ID NO:28 or SEQ ID NO:36, thereby forming a stable duplex.

In one embodiment, an isolated nucleic acid molecule of the presentinvention includes a nucleotide sequence which is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more homologous to the entire length of the nucleotide sequenceshown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:28 or SEQ ID NO:36, or aportion, preferably of the same length, of any of these nucleotidesequences.

A nucleic acid molecule of the invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:28, orSEQ ID NO:36. A nucleic acid includes a nucleotide sequence thatincludes part, or all, of the coding region and extends into either (orboth) the 5′ or 3′ noncoding region. Other embodiments include afragment which includes a nucleotide sequence encoding an amino acidfragment described herein, particularly fragments thereof which are atleast 100 amino acids in length. Fragments also include nucleic acidsequences corresponding to specific amino acid sequences described aboveor fragments thereof. Nucleic acid fragments should not to be construedas encompassing those fragments that may have been disclosed prior tothe invention.

A nucleic acid fragment encoding a “biologically active portion of a B.thetaiotaomicron GAG lyase polypeptide” can be prepared by isolating aportion of the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 22, 28,33, 36 or 38, which encodes a polypeptide having a GAG lyase biologicalactivity (e.g., the biological activities of GAG lyase proteins aredescribed herein), expressing the encoded portion of the B.thetaiotaomicron GAG lyase protein (e.g., by recombinant expression invitro) and assessing the activity of the encoded portion of the B.thetaiotaomicron GAG lyase protein. A nucleic acid fragment encoding abiologically active portion of a B. thetaiotaomicron GAG lyasepolypeptide, may comprise a nucleotide sequence which is greater than300 or more nucleotides in length.

In preferred embodiments, a nucleic acid includes a nucleotide sequencewhich is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900 or more nucleotides in lengthand hybridizes under stringent hybridization conditions to a nucleicacid molecule of SEQ ID NO:1, 3, 5, 7, 9, 22, 28, 33, 36 or 38.

The invention encompasses nucleic acid molecules that differ from thenucleotide sequence shown in SEQ ID NO:1, 3, 5, 7, 9, 22, 28, 33, 36 or38. Such differences can be due to degeneracy of the genetic code (andresult in a nucleic acid which encodes the same B. thetaiotaomicron GAGlyase proteins as those encoded by the nucleotide sequence disclosedherein). In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence which differs, by at least 1, but less than 5, 10, 20, 50,or 100 amino acid residues that shown in SEQ ID NO:2, 4, 6, 8, 10, 23,29, 34, 37 or 39. If alignment is needed for this comparison thesequences should be aligned for maximum homology. “Looped” out sequencesfrom deletions or insertions, or mismatches, are considered differences.

Nucleic acids of the inventor can be chosen for having codons, which arepreferred, or non-preferred, for a particular expression system. E.g.,the nucleic acid can be one in which at least one codon, at preferablyat least 10%, or 20% of the codons has been altered such that thesequence is optimized for expression in E. coli, yeast, human, insect,or CHO cells.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus) and homologs (different locus), or can be nonnaturally occurring. Non-naturally occurring variants can be made bymutagenesis techniques, including those applied to polynucleotides,cells, or organisms. The variants can contain nucleotide substitutions,deletions, inversions and insertions. Variation can occur in either orboth the coding and non-coding regions. The variations can produce bothconservative and non-conservative amino acid substitutions (as comparedin the encoded product).

In a preferred embodiment, the nucleic acid differs from that of SEQ IDNO: 1, 3, 5, 7, 9, 23, 28, 33, 36 or 38, e.g., as follows: by at leastone but less than 10, 20, 30, or 40 nucleotides; at least one but lessthan 1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid.If necessary for this analysis the sequences should be aligned formaximum homology. “Looped” out sequences from deletions or insertions,or mismatches, are considered differences.

Homologs and allelic variants can be identified using methods known inthe art. These variants comprise a nucleotide sequence encoding apolypeptide that is 50%, at least about 55%, typically at least about70-75%, more typically at least about 80-85%, and most typically atleast about 90-95% or more identical to the amino acid sequence shown inSEQ ID NO:2, 4, 6, 8, 10, 23, 29, 34, 37 or 39, or a fragment of thesesequences. Such nucleic acid molecules can readily be identified asbeing able to hybridize under stringent conditions, to the nucleotidesequence encoding the amino acid sequence shown in SEQ ID NO 2, SEQ IDNO:6, SEQ ID NO:29 or SEQ ID NO:37, or a fragment of the sequence.Nucleic acid molecules corresponding to homologs and allelic variants ofthe B. thetaiotaomicron GAG lyase DNAs of the invention can further beisolated by mapping to the same chromosome or locus as the B.thetaiotaomicron GAG lyase gene.

Preferred variants include those that are correlated with a GAG lyaseactivity described herein.

Allelic variants of B. thetaiotaomicron GAG lyase include bothfunctional and non-functional proteins. Functional allelic variants arenaturally occurring amino acid sequence variants of the B.thetaiotaomicron GAG lyase protein within a population that maintain oneor more GAG lyase activity. Functional allelic variants will typicallycontain only conservative substitution of one or more amino acids of SEQID NO:2, 6, 29, or 37, or substitution, deletion or insertion ofnon-critical residues in non-critical regions of the protein. Examplesof functional variants include the M17, Q26, Q23, K169 and D20 variantsdescribed herein (i.e., SEQ ID NOs: 4, 23, 8 10 and 39, respectively),as well as Q23 variant of GAG lyase III (SEQ ID NO:34). Non-functionalallelic variants are naturally-occurring amino acid sequence variants ofthe B. thetaiotaomicron GAG lyase protein within a population that donot have one or more of the GAG lyase activities described herein.Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion, or prematuretruncation of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQID NO:29 or SEQ ID NO:37, or a substitution, insertion, or deletion incritical residues or critical regions of the protein.

Moreover, nucleic acid molecules encoding other B. thetaiotaomicron GAGlyase family members and, thus, which have a nucleotide sequence whichdiffers from the B. thetaiotaomicron GAG lyase sequences of SEQ ID NO:1,SEQ ID NO:5, SEQ ID NO:28 or SEQ ID NO:36 are intended to be within thescope of the invention.

Isolated B. thetaiotaomicron GAG Lyase Polypeptides

In another aspect, the invention features, isolated B. thetaiotaomicronGAG lyase proteins, and fragments thereof, e.g., biologically activeportions thereof. B. thetaiotaomicron GAG lyase protein can be isolatedfrom cells or tissue sources using standard protein purificationtechniques. B. thetaiotaomicron GAG lyase protein or fragments thereofcan be produced by recombinant DNA techniques or synthesized chemically.

Polypeptides of the invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events,alternative RNA splicing events, and alternative translational andpost-translational events. The polypeptide can be expressed in systems,e.g., cultured cells, which result in substantially the samepost-translational modifications present when expressed the polypeptideis expressed in a native cell, or in systems which result in thealteration or omission of post-translational modifications, e.g.,glycosylation or cleavage, present when expressed in a native cell.

In a preferred embodiment, a B. thetaiotaomicron GAG lyase polypeptidehas one or more of the following characteristics: (1) binds a heparinand/or a heparan sulfate; (2) cleaves one or more glycosidic linkages ofa heparin and/or a heparan sulfate; (3) modulates, e.g., increases orreduces, anti-Xa activity and/or anti-IIa activity of a heparin and/or aheparan sulfate; and (4) reduces or eliminates angiogenesis.

In some embodiments, the B. thetaiotaomicron GAG lyase is B.thetaiotaomicron GAG lyase I and the B. thetaiotaomicron GAG lyase I canhave one or more of the following activities: (1) binds a heparin and/orheparan sulfate; (2) cleaves one or more glycosidic linkages of heparinand/or heparan sulfate, e.g., cleaves one or more glycosidic linkages ofsulfated uronic acids, e.g., 2-O uronic acids; cleaves one or moreglycosylic linkages involving sulfated hexosamines, e.g., 6-O sulfatesand/or N-sulfamides; (3) reduces anti-Xa activity and/or anti-IIaactivity of a heparin and/or a heparan sulfate, e.g., as compared to areference standard, e.g., the anti-Xa activity and/or anti-IIa activityof a commercially available heparin or heparan sulfate or of the heparinor heparan sulfate prior to cleavage. In some embodiments, anti-Xaactivity is reduced while anti-IIa activity is maintained. In otherembodiments, anti-Xa activity and anti-IIa activity are reduced.

In some embodiments, the B. thetaiotaomicron GAG lyase is B.thetaiotaomicron GAG lyase II and the B. thetaiotaomicron GAG lyase IIcan have one or more of the following activities: (1) binds a heparinand/or heparan sulfate; (2) cleaves one or more glycosidic linkages ofheparin and/or heparan sulfate, e.g., cleaves one or more glycosidiclinkages of sulfated and undersulfated uronic acids; (3) increasesanti-Xa activity and/or anti-IIa activity of a heparin and/or a heparansulfate, e.g., as compared to a reference standard, e.g., the anti-Xaactivity and/or anti-IIa activity of a commercially available heparin orheparan sulfate or of the heparin or heparan sulfate prior to cleavage.In some embodiments, anti-Xa activity is maintained or increased whileanti-IIa activity is reduced. In other embodiments, anti-IIa activity isincreased while anti-Xa activity is maintained.

In some embodiments, the B. thetaiotaomicron GAG lyase is B.thetaiotaomicron GAG lyase III and the B. thetaiotaomicron GAG lyase IIIcan have one or more of the following activities: (1) binds a heparin,heparan sulfate, chondroitin sulfate and/or dermatin sulfate; (2)cleaves one or more glycosidic linkages of heparin and/or heparansulfate, e.g., cleaves one or more glycosidic linkages of between asulfated hexosamine (e.g., N-sulfated and/or 6-O sulfated) or anunsulfated, but acetylated hexosamine (e.g., HNAc) and a sulfated uronicacid, e.g., a 2-O sulfated uronic acid, or an unsulfated uronic acid;(3) decreases anti-Xa activity and/or anti-IIa activity of a heparinand/or a heparan sulfate, e.g., as compared to a reference standard,e.g., the anti-Xa activity and/or anti-IIa activity of a commerciallyavailable heparin or heparan sulfate or of the heparin or heparansulfate prior to cleavage. In some embodiments, anti-Xa activity isreduced while anti-IIa activity is maintained. In other embodiments,anti-Xa activity and anti-IIa activity are reduced.

In some embodiments, the B. thetaiotaomicron GAG lyase is B.thetaiotaomicron GAG lyase IV and the B. thetaiotaomicron GAG lyase IVcan have one or more of the following activities: (1) binds a heparinand/or heparan sulfate; (2) cleaves one or more glycosidic linkages ofheparin and/or heparan sulfate, e.g., cleaves one or more glycosidiclinkages of low to medium sulfate density, especially linkages involvinga 2-O sulfated uronic acid and adjoining acetylated glucosamine (notcommonly found in most naturally occurring preparations of heparinand/or heparin sulfate); (3) increases anti-Xa activity and/or anti-IIaactivity of a heparin and/or a heparan sulfate, e.g., as compared to areference standard, e.g., the anti-Xa activity and/or anti-IIa activityof a commercially available heparin or heparan sulfate or of the heparinor heparan sulfate prior to cleavage. In some embodiments, anti-Xaactivity is increased while anti-IIa activity is maintained or reduced.In other embodiments, anti-IIa activity is increased while anti-Xaactivity is maintained

In a preferred embodiment, the B. thetaiotaomicron GAG lyase protein, orfragment thereof, differs from the corresponding sequence in SEQ IDNO:2, 4, 6, 8, 10, 23, 29, 34, 37 or 39. In one embodiment, it differsby at least one but by less than 15, 10 or 5 amino acid residues. Inanother, it differs from the corresponding sequence in SEQ ID NO:2, 4,6, 8, 10, 23, 29, 34, 37 or 39 by at least one residue but less than20%, 15%, 10% or 5% of the residues in it differ from the correspondingsequence in SEQ ID NO:2, 4, 6, 8, 10, 23, 29, 34, 37 or 39. (If thiscomparison requires alignment the sequences should be aligned formaximum homology. “Looped” out sequences from deletions or insertions,or mismatches, are considered differences.) The differences are,preferably, differences or changes at a non essential residue or aconservative substitution.

Other embodiments include a protein that contain one or more changes inamino acid sequence, e.g., a change in an amino acid residue which isnot essential for activity. Such B. thetaiotaomicron GAG lyase proteinsdiffer in amino acid sequence from SEQ ID NO:2, 4, 6, 8, 10, 23, 29, 34,37 or 39, yet retain biological activity.

In one embodiment, the protein includes an amino acid sequence at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or morehomologous to SEQ ID NO:2, 4, 6, 8, 10, 23, 29, 34, 37 or 39.

Biologically active portions, in which regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of a native B. thetaiotaomicron GAGlyase protein.

In a preferred embodiment, the B. thetaiotaomicron GAG lyase protein hasan amino acid sequence shown in SEQ ID NOs:2, 4, 6, 8, 10, 23, 29, 34,37 or 39. In other embodiments, the B. thetaiotaomicron GAG lyaseprotein is substantially identical to SEQ ID NOs:2, 4, 6, 8, 10, 23, 29,34, 37 or 39. In yet another embodiment, the B. thetaiotaomicron GAGlyase protein is substantially identical to SEQ ID NOs:2, 4, 6, 8, 10,23, 29, 34, 37 or 39 and retains the functional activity of the proteinof SEQ ID NO:2, 4, 6, 8, 10, 23, 29, 34, 37 or 39, as described indetail in the subsections above.

In another aspect, the invention provides B. thetaiotaomicron GAG lyasechimeric or fusion proteins. As used herein, a B. thetaiotaomicron GAGlyase “chimeric protein” or “fusion protein” includes a B.thetaiotaomicron GAG lyase polypeptide linked to a non-B.thetaiotaomicron GAG lyase polypeptide. A “non-B. thetaiotaomicron GAGlyase polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein which is not substantially homologous to theB. thetaiotaomicron GAG lyase protein, e.g., a protein which isdifferent from the B. thetaiotaomicron GAG lyase protein and which isderived from the same or a different organism. The B. thetaiotaomicronGAG lyase polypeptide of the fusion protein can correspond to all or aportion e.g., a fragment described herein, of a B. thetaiotaomicron GAGlyase amino acid sequence. In a preferred embodiment, a B.thetaiotaomicron GAG lyase fusion protein includes at least one (or two)biologically active portion of a B. thetaiotaomicron GAG lyase protein.The non-B. thetaiotaomicron GAG lyase polypeptide can be fused to theN-terminus or C-terminus of the B. thetaiotaomicron GAG lyasepolypeptide.

The fusion protein can include a moiety which has a high affinity for aligand. For example, the fusion protein can be a GST-B. thetaiotaomicronGAG lyase fusion protein in which the B. thetaiotaomicron GAG lyasesequences are fused to the C-terminus of the GST sequences. Such fusionproteins can facilitate the purification of recombinant B.thetaiotaomicron GAG lyase. Alternatively, the fusion protein can be aB. thetaiotaomicron GAG lyase protein containing a heterologous signalsequence at its N-terminus. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of B. thetaiotaomicron GAG lyase canbe increased through use of a heterologous signal sequence. Moreover,the B. thetaiotaomicron GAG lyase-fusion proteins of the invention canbe used as immunogens to produce anti-B. thetaiotaomicron GAG lyaseantibodies in a subject, to purify B. thetaiotaomicron GAG lyase ligandsand in screening assays to identify molecules which inhibit theinteraction of B. thetaiotaomicron GAG lyase with a B. thetaiotaomicronGAG lyase substrate.

Expression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). A B. thetaiotaomicron GAGlyase-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the B.thetaiotaomicron GAG lyase protein.

In another aspect, the invention also features a variant of a B.thetaiotaomicron GAG lyase polypeptide, e.g., which functions as anagonist (mimetics). Variants of the B. thetaiotaomicron GAG lyaseproteins can be generated by mutagenesis, e.g., discrete point mutation,the insertion or deletion of sequences or the truncation of a B.thetaiotaomicron GAG lyase protein. An agonist of the B.thetaiotaomicron GAG lyase proteins can retain substantially the same,or a subset, of the biological activities of the naturally occurringform of a B. thetaiotaomicron GAG lyase protein.

Variants of a B. thetaiotaomicron GAG lyase protein can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of a B. thetaiotaomicron GAG lyase protein for agonist activity.Variants of a B. thetaiotaomicron GAG lyase I include the M17 variant asshown in SEQ ID NO:4 and the Q26 variant as shown in SEQ ID NO:23.Variants of a B. thetaiotaomicron GAG lyase II include the Q23 variantas shown in SEQ ID NO:8 and the K169 variant as shown in SEQ ID NO:10.Variants of B. thetaiotaomicron GAG lyase III include the Q23 variantshown in SEQ ID:34. Variants of B. thetaiotaomicron GAG lyase IIIinclude the D20 variant shown in SEQ ID:39.

Libraries of fragments e.g., N terminal, C terminal, or internalfragments, of a B. thetaiotaomicron GAG lyase protein coding sequencecan be used to generate a variegated population of fragments forscreening and subsequent selection of variants of a B. thetaiotaomicronGAG lyase protein. Variants in which a cysteine residues is added ordeleted or in which a residue which is glycosylated is added or deletedare particularly preferred.

Methods for screening gene products of combinatorial libraries made bypoint mutations or truncation, and for screening cDNA libraries for geneproducts having a selected property are known in the art. Such methodsare adaptable for rapid screening of the gene libraries generated bycombinatorial mutagenesis of B. thetaiotaomicron GAG lyase proteins.Recursive ensemble mutagenesis (REM), a new technique which enhances thefrequency of functional mutants in the libraries, can be used incombination with the screening assays to identify B. thetaiotaomicronGAG lyase variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).

Cell based assays can be exploited to analyze a variegated B.thetaiotaomicron GAG lyase library. For example, a library of expressionvectors can be transfected into a cell line, e.g., a cell line, whichordinarily responds to B. thetaiotaomicron GAG lyase in asubstrate-dependent manner The transfected cells are then contacted withB. thetaiotaomicron GAG lyase and the effect of the expression of themutant on the activity of the B. thetaiotaomicron GAG lyase substratecan be detected, e.g., by measuring cleavage of heparin or heparansulfate. Plasmid DNA can then be recovered from the cells which scorefor inhibition, or alternatively, potentiation of signaling by the B.thetaiotaomicron GAG lyase substrate, and the individual clones furthercharacterized.

In another aspect, the invention features a method of making a fragmentor analog of a naturally occurring B. thetaiotaomicron GAG lyasepolypeptide. The method includes: altering the sequence, e.g., bysubstitution or deletion of one or more residues, of a B.thetaiotaomicron GAG lyase polypeptide, e.g., altering the sequence of anon-conserved region, or a domain or residue described herein, andtesting the altered polypeptide for the desired activity, e.g., asdescribed above.

Recombinant Expression Vectors, Host Cells and Genetically EngineeredCells

In another aspect, the invention includes, vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidedescribed herein. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked and can include a plasmid, cosmid or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors include, e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses.

A vector can include a B. thetaiotaomicron GAG lyase nucleic acid in aform suitable for expression of the nucleic acid in a host cell.Preferably the recombinant expression vector includes one or moreregulatory sequences operatively linked to the nucleic acid sequence tobe expressed. The term “regulatory sequence” includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Regulatory sequences include those which direct constitutiveexpression of a nucleotide sequence, as well as tissue-specificregulatory and/or inducible sequences. The design of the expressionvector can depend on such factors as the choice of the host cell to betransformed, the level of expression of protein desired, and the like.The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or polypeptides, including fusionproteins or polypeptides, encoded by nucleic acids as described herein(e.g., B. thetaiotaomicron GAG lyase proteins, mutant forms of B.thetaiotaomicron GAG lyase proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of B. thetaiotaomicron GAG lyase proteins in prokaryotic oreukaryotic cells. For example, polypeptides of the invention can beexpressed in E. coli, insect cells (e.g., using baculovirus expressionvectors), yeast cells or mammalian cells. Suitable host cells arediscussed further in Goeddel, (1990) Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant protein.

Purified fusion proteins can be used in B. thetaiotaomicron GAG lyaseactivity assays, (e.g., direct assays or competitive assays described indetail below), or to generate antibodies specific for B.thetaiotaomicron GAG lyase proteins. In a preferred embodiment, a fusionprotein expressed in a retroviral expression vector of the presentinvention can be used to infect bone marrow cells which are subsequentlytransplanted into irradiated recipients. The pathology of the subjectrecipient is then examined after sufficient time has passed (e.g., sixweeks).

To maximize recombinant protein expression in E. coli is to express theprotein in a host bacteria with an impaired capacity to proteolyticallycleave the recombinant protein (Gottesman, S., (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.119-128). Another strategy is to alter the nucleic acid sequence of thenucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

The B. thetaiotaomicron GAG lyase expression vector can be a yeastexpression vector, a vector for expression in insect cells, e.g., abaculovirus expression vector or a vector suitable for expression inmammalian cells.

When used in mammalian cells, the expression vector's control functionscan be provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40.

In another embodiment, the promoter is an inducible promoter, e.g., apromoter regulated by a steroid hormone, by a polypeptide hormone (e.g.,by means of a signal transduction pathway), or by a heterologouspolypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and“Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy9:983).

In still another embodiment, the recombinant mammalian expression vectoris capable of directing expression of the nucleic acid preferentially ina particular cell type (e.g., tissue-specific regulatory elements areused to express the nucleic acid). Non-limiting examples of suitabletissue-specific promoters include the albumin promoter (liver-specific;Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters(Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particularpromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740;Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al.(1985) Science 230:912-916), and mammary gland-specific promoters (e.g.,milk whey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example, the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. Regulatory sequences (e.g., viralpromoters and/or enhancers) operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus.

Another aspect the invention provides a host cell which includes anucleic acid molecule described herein, e.g., a B. thetaiotaomicron GAGlyase nucleic acid molecule within a recombinant expression vector or aB. thetaiotaomicron GAG lyase nucleic acid molecule containing sequenceswhich allow it to homologously recombine into a specific site of thehost cell's genome. The terms “host cell” and “recombinant host cell”are used interchangeably herein. Such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a B.thetaiotaomicron GAG lyase protein can be expressed in bacterial cells(such as E. coli), insect cells, yeast or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells). Other suitable hostcells are known to those skilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

A host cell of the invention can be used to produce (i.e., express) a B.thetaiotaomicron GAG lyase protein. Accordingly, the invention furtherprovides methods for producing a B. thetaiotaomicron GAG lyase proteinusing the host cells of the invention. In one embodiment, the methodincludes culturing the host cell of the invention (into which arecombinant expression vector encoding a B. thetaiotaomicron GAG lyaseprotein has been introduced) in a suitable medium such that a B.thetaiotaomicron GAG lyase protein is produced. In another embodiment,the method further includes isolating a B. thetaiotaomicron GAG lyaseprotein from the medium or the host cell.

In another aspect, the invention features, a cell or purifiedpreparation of cells which include a B. thetaiotaomicron GAG lyasetransgene, or which otherwise misexpress B. thetaiotaomicron GAG lyase.The cell preparation can consist of human or non-human cells, e.g.,rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. Inpreferred embodiments, the cell or cells include a B. thetaiotaomicronGAG lyase transgene, e.g., a heterologous form of a B. thetaiotaomicronGAG lyase, e.g., a gene derived from humans (in the case of a non-humancell). The B. thetaiotaomicron GAG lyase transgene can be misexpressed,e.g., overexpressed or underexpressed. In other preferred embodiments,the cell or cells include a gene that mis-expresses an endogenous B.thetaiotaomicron GAG lyase, e.g., a gene the expression of which isdisrupted, e.g., a knockout. Such cells can serve as a model forstudying disorders that are related to mutated or mis-expressed B.thetaiotaomicron GAG lyase alleles or for use in drug screening.

In another aspect, the invention features, a human cell, e.g., ahematopoietic stem cell, transformed with nucleic acid which encodes asubject B. thetaiotaomicron GAG lyase polypeptide.

Also provided are cells in which a B. thetaiotaomicron GAG lyase isunder the control of a regulatory sequence that does not normallycontrol the expression of the endogenous B. thetaiotaomicron GAG lyasegene. The expression characteristics of an endogenous gene within acell, e.g., a cell line or microorganism, can be modified by inserting aheterologous DNA regulatory element into the genome of the cell suchthat the inserted regulatory element is operably linked to theendogenous B. thetaiotaomicron GAG lyase gene. For example, anendogenous B. thetaiotaomicron GAG lyase gene which is“transcriptionally silent,” e.g., not normally expressed, or expressedonly at very low levels, may be activated by inserting a regulatoryelement which is capable of promoting the expression of a normallyexpressed gene product in that cell. Techniques such as targetedhomologous recombinations, can be used to insert the heterologous DNA asdescribed in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667,published in May 16, 1991.

Uses

As described herein, the B. thetaiotaomicron GAG lyase molecules of theinvention are useful in many applications including, but not limitedto: 1) characterization of GAGs such as heparins and heparan sulfates(and, to some extent, chondroitin sulfate and dermatan sulfate) in termsof chemical composition (di-, tri-, tetra-, penta-, hexa-, octa-, and/ordeca-oligosaccharides); 2) characterization of a pharmaceuticalformulation of GAGs such as a formulation of heparin or a heparansulfate (and, to some extent, chondroitin sulfate and dermatan sulfate);3) fractionation of a GAG such as a heparin and a heparan sulfate (and,to some extent, chondroitin sulfate and dermatan sulfate) into both itschemical constituents as well as into smaller fragments of definedlength, sequence, and potential bioactivities; 4) in vitroneutralization of the anticoagulant activity (anti-Xa) of a heparin or aheparan sulfate; 5) in vitro modulation of antithrombotic activity (antiIIa); 6) identification of the presence and purity of a particular GAGsuch as a heparin or a heparan sulfate in a sample; 7) determination ofthe composition of a GAG in a sample; 8) determination of the sequenceof di-, tetra-, hexa-, octa- and deca-saccharide units in a particularheparin or heparan sulfate; 9) use as an additional analytic tool forchemical analysis using techniques such as mass spectrometry, NMRspectroscopy, gel electrophoresis, capillary electrophoresis, HPLC, andion-pair HPLC; 10) for cleaving a particular GAG such as a heparin orheparan sulfate that comprises at least two disaccharide units; 11) forinhibiting angiogenesis, e.g., through administration to a subject inneed thereof an effective amount of a composition (e.g., apharmaceutical composition) containing B. thetaiotaomicron GAG lyasemolecules; 12) for treating cancer through the administration to asubject a composition (e.g., a pharmaceutical composition) containing B.thetaiotaomicron GAG lyase molecules; 13) inhibiting cellularproliferation through the administration to a subject in need thereof aneffective amount of a composition (e.g., a pharmaceutical composition)containing B. thetaiotaomicron GAG lyase molecules for inhibitingcellular proliferation; 14) for ex vivo neutralization of the anti-Xaactivity of a preparation (e.g., a pharmaceutical preparation) of aheparin or a heparan sulfate previously administered to a subject forthe inhibition of coagulation; 15) for in vivo neutralization of theanti-Xa activity of preparation (e.g., a pharmaceutical preparation) ofa heparin or a heparan sulfate through administration to a subject inneed of such neutralization (e.g., a subject to whom a pharmaceuticalpreparation of a heparin or a heparan sulfate had previously beenadministered); 16) for ex vivo neutralization of the anti-Ha activity ofa preparation (e.g., pharmaceutical preparation) of a heparin or heparansulfate previously administered to a subject for the inhibition ofthrombosis; or 17) for in vivo neutralization of the anti-IIa activityof preparation (e.g., a pharmaceutical preparation) of a heparin or aheparan sulfate through administration to a subject in need in need ofsuch neutralization (e.g., a subject to whom a pharmaceuticalpreparation of a heparin or heparan sulfate had previously beenadministered).

Characterization and Sequencing of GAGs

Methods described herein can be used, e.g., for analyzingpolysaccharides such as GAGs, (e.g., a mixed population ofpolysaccharides), e.g., to define the structural signature and/oractivity of a polysaccharides (e.g., a mixed population ofpolysaccharides), by contacting the polysaccharide with a B.thetaiotaomicron GAG lyase molecule. A structural signature, as usedherein, refers to information regarding, e.g., the identity and numberthe mono- and di-saccharide building blocks of a polysaccharide,information regarding the physiochemical properties such as the overallcharge (also referred to as the “net charge” or “total charge”), chargedensity, molecular size, charge to mass ratio and the presence ofiduronic and/or glucuronic acid content as well as the relationshipsbetween the mono- and di-saccharide building blocks, and active sitesassociated with these building blocks, inter alia. The structuralsignature can be provided by determining one or more primary outputsthat include the following: the presence or the amount of one or morecomponent saccharides or disaccharides; as used herein, “componentsaccharides” refers to the saccharides that make up the polysaccharide.Component saccharides can include monosaccharides, disaccharides,trisaccharides, etc., and can also include sugars normally found innature as well as non-natural and modified sugars, e.g., that result dueto production, processing and/or purification; the presence or theamount of one or more block components, wherein a “block component” ismade up of more than one saccharide or polysaccharide; and the presenceor amount of one or more modified saccharides, wherein a modifiedsaccharide is one present in a starting material used to make apreparation but which is altered in the production of the preparation,e.g., a saccharide modified by cleavage. “Sequence” with respect topolysaccharides refers to the linear arrangement of covalently linkedcomponent saccharides, and can be determined by methods known in theart, e.g., the methods disclosed herein and in PCT Publication Nos: WO00/65521, WO 02/23190, and WO 04/055491; U.S. Publication Nos:20030191587 and 20040197933; Venkataraman (1999); Shriver et al.(2000a); Shriver et al. (2000b); and Keiser et al. (2001); the entireteachings of which are incorporated herein by reference. “Positioning ofthe active site” refers to a correlation between a certain componentpolysaccharide and a given activity.

In one embodiment, the invention provides, methods of evaluating apolysaccharide mixture, e.g., a heterogeneous population of HLGAGs, byevaluating one or more parameters related to a structural signaturespecies described herein. Such parameters can include the presence, sizedistribution, or quantity of a structural signature disclosed herein.The structural signature can be one or more of the following:

In a preferred embodiment, the structural signature is determined by oneor more methods chosen from the group consisting of MALDI-MS, ESI-MS,CE, HPLC, FPLC, fluorometry, ELISA, chromogenic assays such as reversephase column chromatography (e.g., HPLC), colorimetric assays, NMR andother spectroscopic techniques.

The polysaccharide composition is digested, incompletely or completelydigested, with one or more B. thetaiotaomicron GAG lyase molecule. Thecomposition can further be digested with one or more HLGAG degradingenzyme. Examples of other HLGAG degrading enzymes include: heparinase I,heparinase II, heparinase III, heparinase IV, heparanase,D-glucuronidase, L-iduronidase and functionally active variants andfragments thereof. Various HLGAG degrading enzymes, and variants andfragments thereof, are known and described, e.g., in U.S. Pat. Nos.5,569,600; 5,389,539; 5,830,726; 5,714,376; 5,919,693; 5,681,733 and6,869,789; and U.S. Patent Publications Nos: 20030099628; 20030303301;and 20010565375, the contents of which are incorporated herein byreference.

The methods described herein can further include: providing ordetermining a first structural signature by contacting a batch of apolysaccharide (e.g., a heterogenous population of polysaccharides) witha B. thetaiotaomicron GAG lyase molecule or molecules; providing ordetermining a second structural signature of a different batch of apolysaccharide (e.g., a heterogenous population of polysaccharides) bycontacting the batch with a B. thetaiotaomicron GAG lyase molecule ormolecules; and comparing the first and second structural determinationsto determine if one or more of the batches has a structuraldetermination associated with a particular property. The methods canfurther include selecting or discarding a batch of the polysaccharidedepending on its structural determination.

In other embodiments, a batch of a polysaccharide (e.g., a heterogenouspopulation of polysaccharides) can be analyzed by comparing one or morestructural signature of the polysaccharide obtained by contacting thepolysaccharide with one or more B. thetaiotaomicron GAG lyase moleculesto a reference standard. The reference standard can be, e.g., apreselected range or level and/or the absence or presence of astructural signature present in a mixed population of polysaccharides,e.g., a commercially available population of polysaccharides such asenoxaparin (Lovenox™); dalteparin (Fragmin™); certoparin (Sandobarin™);ardeparin (Normiflo™); nadroparin (Fraxiparin™); parnaparin (Fluxum™);reviparin (Clivarin™); tinzaparin (Innohep™ or Logiparin™), orFondaparinux (Arixtra™) that has been digested with the B.thetaiotaomicron GAG lyase molecule or molecules.

The B. thetaiotaomicron GAG lyase molecules can also be used todetermine a reference standard for a drug by analyzing a compositioncontacted with a B. thetaiotaomicron GAG lyase molecule or molecules anddetermining the bioequivalence and/or bioavailability of one or more ofthe components in the mixture. As used herein, “bioequivalence” means“the absence of a significant difference in the rate and extent to whichan active ingredient or active moiety in pharmaceutical equivalents orpharmaceutical alternatives becomes available at the site of drug actionwhen administered at the same molar dose under similar conditions.”

Production of Fractionated HLGAG Preparations

The B. thetaiotaomicron GAG lyase molecules described herein can be usedto produce polysaccharides (e.g., fractionated heparin or heparansulfate), e.g., having desired properties, e.g., desired activitiesand/or reduced undesired properties, e.g., undesired side effects. Asused herein, “desired activities” refers to those activities that arebeneficial for a given indication, e.g., a positive patient reaction asdefined herein, inter alia. An “undesirable activity” may include thoseactivities that are not beneficial for a given indication, e.g., anegative patient reaction, as defined herein, inter alia. A givenactivity may be a desired activity for one indication, and an undesiredactivity for another, such as anti-IIa activity, which while undesirablefor certain indications, is desirable in others, notably acute or traumasituations. Thus, the invention relates to methods for designingheparins, LMWHs or synthetic heparins with ideal product profilesincluding, but not limited to such features as high activity, e.g., highanti-Xa and/or anti-IIa activity, reduced activity, e.g., reducedanti-Xa and/or anti-IIa activity, well characterized, neutralizable,lower side effects including reduced HIT, attractive pharmacokinetics,and/or reduced PF4 binding.

Fractionated heparins can be designed, e.g., by contacting compositionthat includes a mixed population of polysaccharides, such asglycosaminoglycans (GAGs), HLGAGs, UFH, FH, LMWHs, or synthetic heparinsincluding but not limited to enoxaparin (Lovenox™); dalteparin(Fragmin™); certoparin (Sandobarin™); ardeparin (Normiflo™); nadroparin(Fraxiparin™); parnaparin (Fluxum™); reviparin (Clivarin™); tinzaparin(Innohep™ or Logiparin™), or Fondaparinux (Arixtra™) with a B.thetaiotaomicron GAG lyase.

In some embodiments, a fractionated heparin preparation having reducedanti-Xa and/or anti-IIa activity is prepared by contacting a heparinwith a B. thetaiotaomicron GAG lyase I and/or B. thetaiotaomicron GAGlyase III molecule. In some embodiments, anti-Xa activity is reducedwhile anti-IIa activity is maintained In other embodiments, anti-Xaactivity and anti-IIa activity are reduced. Heparins having reducedanti-Xa and/or anti-IIa activity can be used, e.g., as a carrier todeliver an agent, e.g., a diagnostic, prophylactic or therapeutic agent.The heparin molecule can be linked to the agent. Active agents caninclude a therapeutic or prophylactic polypeptide, nucleic acid, smallmolecule, lipid/glycolipids, etc. In one embodiment, the active agent isa therapeutic polypeptide selected from the group consisting of insulin,proinsulin, human growth hormone, interferon, α-1 proteinase inhibitor,alkaline phosphotase, angiogenin, cystic fibrosis transmembraneconductance regulator, extracellular superoxide dismutase, fibrinogen,glucocerebrosidase, glutamate decarboxylase, human serum albumin, myelinbasic protein, soluble CD4, lactoferrin, lactoglobulin, lysozyme,lactoalbumin, erythropoietin, tissue plasminogen activator, antithrombinIII, prolactin, and α1-antitrypsin. The therapeutic or prophylacticpolypeptide can be an active derivative or fragment of suchpolypeptides. The active agent can also be, but is not limited to one ormore of: parathyroid hormone and derivatives and fragments thereof,erythropoietin, epoetin beta, gene activated erythropoietin, secondgeneration EPO, novel erythropoiesis stimulating protein, insulinlispro, insulin (bovine), insulin, insulin aspart, insulin analogue,Calcitonin, Theraccine, becaplermin (recombinant human platelet derivedgrowth factor-BB), trafermin, human growth hormone-releasing factor,BMP-7, PEG asparaginase, dornase alpha, alglucerase, agalsidase-beta,dornase alpha, agalsidase-alfa, streptokinase, teneteplase, reteplase,alteplase, pamiteplase, Rh factor VIII, Rh FVIIa, Factor IX (Human),Factor IX (complex), HGH, Somatrem/somatropin, anti-CD33-calicheamicinconjugate, Edrecolomab, rituxumab, daclizumab, trastuzumab, sulesomab,abciximab, infliximab, muromonab-CD3, palivizumab, alemtuzumab,basiliximab, oprelvekin, gemtuzamab ozogamicin, ibritumomab tiuxetan,sulesomab, palivizumab, interleukin-2, celmoleukin (rIL-2), interferonalfacon-1, interferon alpha, interferon alpha+ribavirin, peg interferonalpha-2a, interferon alpha-2b, interferon alpha 3n, interferon beta-1a,interferon beta, interferon beta 1b, interferon gamma, interferongamma-1b, filgrastim, sargramostim, lenograstim, molgramostim,mirimostim, nartograstim, oprelvekin, peptide tyrosin-tyrosin (PYY),apolipoprotein A-IV, leptin, melanocortin, amylin, orexin, adiponectin,and ghrelin. In one embodiment, the active agent is an activepolypeptide, e.g., a therapeutic or prophylactic polypeptide, and thepolypeptide has a molecular weight of less than 150 kDa, more preferablyless than 100 kDa, and more preferably less than 50 kDa. In oneembodiment, the active agent is an active polypeptide, e.g., atherapeutic or prophylactic polypeptide, and the polypeptide has amolecular weight of about 500Da-5 kDa, 5 to 10 kDa, 10 to 30 kDa, 18 to35 kDa, 30 to 50 kDa, 50 to 100 kDa, 100 to 150 kDa. In one embodiment,the active polypeptide is insulin or an active fragments or derivativesthereof. In another embodiment, the active polypeptide is human growthhormone or an active fragment or derivative thereof. In yet anotherembodiment, the active polypeptide is interferon. In other embodiments,the heparin molecule is linked to an inactive agent. Examples ofinactive agents include biological probes or contrast agents forimaging. In another embodiment, the active agent can be a small moleculedrug, e.g., a small molecule drug currently available for therapeutic,diagnostic, or prophylactic use, or a drug in development. In someembodiments, the active agent is linked to one or more heparin moleculesin the formulation. As an example, small molecule drugs, andprotein-based drugs may be linked to heparin molecule for delivery viaknown chemistries such as EDC, CNBH₄/DMSO/Acetic Acid, etc.

The invention also relates to fractionated heparin preparations havingincreased anti-Xa and/or anti-IIa activity prepared by contacting aheparin with a B. thetaiotaomicron GAG lyase II and/or B.thetaiotaomicron GAG lyase IV molecule. Such preparation can be used,e.g., to treat or prevent a disease associated with coagulation, such asthrombosis, cardiovascular disease, vascular conditions or atrialfibrillation; migraine, atherosclerosis; an inflammatory disorder, suchas autoimmune disease or atopic disorders; obesity or excess adipose, anallergy; a respiratory disorder, such as asthma, emphysema, adultrespiratory distress syndrome (ARDS), cystic fibrosis, or lungreperfusion injury; a cancer or metastatic disorder, e.g., lipomas;diabetes; an angiogenic disorder, such as neovascular disorders of theeye, osteoporosis, psoriasis, arthritis, Alzheimer's, a subject toundergo, undergoing or having undergone surgical procedure, organtransplant, orthopedic surgery, treatment for a fracture, e.g., a hipfracture, hip replacement, knee replacement, percutaneous coronaryintervention (PCI), stent placement, angioplasty, coronary artery bypassgraft surgery (CABG).

Pharmaceutical Compositions

The B. thetaiotaomicron GAG lyase molecules, as well as heparinmolecules prepared by cleavage with the B. thetaiotaomicron GAG lyasemolecules can be incorporated into pharmaceutical compositions. Suchcompositions typically include a pharmaceutically acceptable carrier. Asused herein the language “pharmaceutically acceptable carrier” includessolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Alternatively, the pharmaceutical composition can be used to treat asample (e.g., blood in a bioreactor, e.g., to deheparinize blood) beforethe sample is introduced into a subject.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Therapeutic Applications

The B. thetaiotaomicron GAG lyase molecules can act as novel diagnosticand therapeutic agents for controlling one or more of cellularproliferative and/or differentiative disorders, e.g., by preventing orinhibiting angiogenesis of cells otherwise exhibiting or otherwiseassociated with unwanted proliferation and/or differentiation. Examplesof cellular and/or differentiative disorders include: diabetes;arthritis, e.g., rheumatoid arthritis; ocular disorders, e.g., ocularneovascularization, diabetic retinopathy, neovascular glaucoma, retinalfibroplasias, uveitis, eye disorders associated with irisneovascularization; and cancer, e.g., carcinoma, sarcoma, metastaticdisorders or hematopoietic neoplastic disorders, e.g., leukemias.

As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth.Examples of such cells include cells having an abnormal state orcondition characterized by rapidly proliferating cell growth.Hyperproliferative and neoplastic disease states may be categorized aspathologic, i.e., characterizing or constituting a disease state, or maybe categorized as non-pathologic, i.e., a deviation from normal but notassociated with a disease state. The term is meant to include all typesof cancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. “Pathologichyperproliferative” cells occur in disease states characterized bymalignant tumor growth. Examples of non-pathologic hyperproliferativecells include proliferation of cells associated with wound repair.

The terms “cancer” or “neoplasms” include malignancies of the variousorgan systems, such as affecting lung, breast, thyroid, lymphoid,gastrointestinal, and genito-urinary tract, as well as adenocarcinomaswhich include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer and/or testicular tumors, non-small cellcarcinoma of the lung, cancer of the small intestine and cancer of theesophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin. A hematopoietic neoplastic disorder can arise frommyeloid, lymphoid or erythroid lineages, or precursor cells thereof.Preferably, the diseases arise from poorly differentiated acuteleukemias, e.g., erythroblastic leukemia and acute megakaryoblasticleukemia. Additional exemplary myeloid disorders include, but are notlimited to, acute promyeloid leukemia (APML), acute myelogenous leukemia(AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L.(1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignanciesinclude, but are not limited to acute lymphoblastic leukemia (ALL) whichincludes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia(CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease. The data obtained from the cell culture assaysand animal studies can be used in formulating a range of dosage for usein humans. The dosage of such compounds lies preferably within a rangeof circulating concentrations that include the ED₅₀ with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose can be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

In another embodiment, the B. thetaiotaomicron GAG lyase molecules,e.g., the B. thetaiotaomicron GAG lyase I and/or the B. thetaiotaomicronGAG lyase III molecules, can act as prophylactic or therapeutic agentsfor controlling heparin-associated disorders. Examples of such disordersinclude, but are not limited to, heparin-induced anticoagulation and/orangiogenesis. Thus, the B. thetaiotaomicron GAG lyase molecules, e.g.,the B. thetaiotaomicron GAG lyase I and/or the B. thetaiotaomicron GAGlyase III molecules, can be used to reduce or eliminate (e.g.,neutralize) one or more anticoagulation and/or antithrombotic propertiesof heparin and/or heparan sulfate, e.g., during or after surgery. Inother embodiments, the B. thetaiotaomicron GAG lyase molecules, e.g.,the B. thetaiotaomicron GAG lyase I and/or the B. thetaiotaomicron GAGlyase III molecules, can be used to deheparinized blood, e.g., in abioreactor, e.g., a bioreactor used in heart-lung and/or kidneydialysis.

The B. thetaiotaomicron GAG lyase molecules described herein can also beused to design fractionated GAG preparations, e.g., heparin and/orheparan sulfate preparations. Such fractionated HLGAG preparations mayhave many therapeutic utilities. For instance, it is known that HLGAGcompositions are useful for preventing and treating dementia, such asAlzheimer's disease, coagulation, angiogenesis, thrombotic disorders,cardiovascular disease, vascular conditions, atherosclerosis,respiratory disorders, circulatory shock and related disorders, as wellas inhibiting cancer cell growth and metastasis. Each of these disordersis well-known in the art and is described, for instance, in Harrison'sPrinciples of Internal Medicine (McGraw Hill, Inc., New York), which isincorporated by reference. The use of HLGAG compositions in varioustherapeutic methods is described and summarized in Huang, J. andShimamura, A., Coagulation Disorders, 12, 1251-1281 (1998).

The fractionated HLGAG preparations can be used, e.g., to treat orprevent a disorder where increased presence of active FGF, e.g., aFGFand/or bFGF, is desirable.

The HLGAG preparations are useful for treating or preventing disordersassociated with coagulation. When an imbalance in the coagulationpathway shifts towards excessive coagulation, the result is thedevelopment of thrombotic tendencies, which are often manifested asheart attacks, strokes, deep venous thrombosis, acute coronary syndromes(ACS) such as unstable angina, and myocardial infarcts. A “diseaseassociated with coagulation” as used herein refers to a conditioncharacterized by local inflammation which can result from aninterruption or reduction in the blood supply to a tissue which mayoccur, for instance, as a result of blockage of a blood vesselresponsible for supplying blood to the tissue such as is seen formyocardial or cerebral infarction or peripheral vascular disease, or asa result of emboli formation associated with conditions such as atrialfibrillation or deep venous thrombosis. Coagulation disorders include,but are not limited to, cardiovascular disease and vascular conditionssuch as cerebral ischemia. It is particularly useful to treat disorderssuch as myocardial infarction and ACS with, e.g., a polysaccharide bypulmonary delivery because of the fast absorption and action of thisdelivery system.

The fractionated HLGAG preparations are useful for treatingcardiovascular disease. Cardiovascular diseases include, but are notlimited to, acute myocardial infarction, ACS, e.g., unstable angina, andatrial fibrillation. Myocardial infarction is a disease state whichsometimes occurs with an abrupt decrease in coronary blood flow thatfollows a thrombotic occlusion of a coronary artery previously narrowedby atherosclerosis. Such injury may be produced or facilitated byfactors such as cigarette smoking, hypertension, and lipid accumulation.Acute angina is due to transient myocardial ischemia. This disorder isusually associated with a heaviness, pressure, squeezing, smothering, orchoking feeling below the sternum. Episodes are usually caused byexertion or emotion, but can occur at rest.

Atrial fibrillation is a common form of arrhythmia generally arising asa result of emotional stress or following surgery, exercise, or acutealcoholic intoxication. Persistent forms of atrial fibrillationgenerally occur in patients with cardiovascular disease. Atrialfibrillation is characterized by disorganized atrial activity withoutdiscrete P waves on the surface ECG. This disorganized activity can leadto improper blood flow in the atrium and thrombus formation. Thesethrombi can embolize, resulting in cerebral ischemia and otherdisorders.

Persons undergoing surgery, anesthesia and extended periods of bed restor other inactivity are often susceptible to a condition known as deepvenous thrombosis, or DVT, which is a clotting of venous blood in thelower extremities and/or pelvis. This clotting occurs due to the absenceof muscular activity in the lower extremities required to pump thevenous blood (stasis), local vascular injury or a hypercoaguble state.The condition can be life-threatening if a blood clot migrates to thelung, resulting in a “pulmonary embolus” or otherwise interferes withcardiovascular circulation. One method of treatment involvesadministration of an anti-coagulant.

The fractionated HLGAG preparations can be used for the treatment ofcardiovascular disorders alone or in combination with other therapeuticagents for reducing the risk of a cardiovascular disease or for treatingthe cardiovascular disease. Other therapeutic agents include, but arenot limited to, anti-inflammatory agents, anti-thrombotic agents,anti-platelet agents, fibrinolytic agents, lipid reducing agents, directthrombin inhibitors, anti-Xa inhibitors, anti-IIa inhibitors,glycoprotein IIb/IIIa receptor inhibitors and direct thrombin inhibitorssuch as hirudin, hirugen, Angiomax, agatroban, PPACK, thrombin aptamers.

The HLGAG preparations are also useful for treating vascular conditions.Vascular conditions include, but are not limited to, disorders such asdeep venous thrombosis, peripheral vascular disease, cerebral ischemia,including stroke, and pulmonary embolism. A cerebral ischemic attack orcerebral ischemia is a form of ischemic condition in which the bloodsupply to the brain is blocked. This interruption or reduction in theblood supply to the brain may result from a variety of causes, includingan intrinsic blockage or occlusion of the blood vessel itself, aremotely originated source of occlusion, decreased perfusion pressure orincreased blood viscosity resulting in inadequate cerebral blood flow,or a ruptured blood vessel in the subarachnoid space or intracerebraltissue.

The HLGAG preparations are useful for treating cerebral ischemia.Cerebral ischemia may result in either transient or permanent deficitsand the seriousness of the neurological damage in a patient who hasexperienced cerebral ischemia depends on the intensity and duration ofthe ischemic event. A transient ischemic attack is one in which theblood flow to the brain is interrupted only briefly and causes temporaryneurological deficits, which often are clear in less than 24 hours.Symptoms of TIA include numbness or weakness of face or limbs, loss ofthe ability to speak clearly and/or to understand the speech of others,a loss of vision or dimness of vision, and a feeling of dizziness.Permanent cerebral ischemic attacks, also called stroke, are caused by alonger interruption or reduction in blood flow to the brain resultingfrom either a thrombus or embolism. A stroke causes a loss of neuronstypically resulting in a neurologic deficit that may improve but thatdoes not entirely resolve.

Thromboembolic stroke is due to the occlusion of an extracranial orintracranial blood vessel by a thrombus or embolus. Because it is oftendifficult to discern whether a stroke is caused by a thrombosis or anembolism, the term “thromboembolism” is used to cover strokes caused byeither of these mechanisms.

The rapid absorption of HLGAGs, such as UFH or LMWH, after inhalationcan be very valuable in the treatment of venous thromboembolism.Intravenous administration of UFH has been used widely for treatment ofvenous thromboembolism in combination with oral warfarin. Due to theimproved efficacy and reduced risks, however, LMWHs have beenincreasingly used as an alternative to intravenous UFH in treatment ofvenous thromboembolism. It has been established that efficacy of heparintherapy depends on achieving critical therapeutic levels (e.g., ofvalues of anti-factor Xa or anti-factor IIa activity) within the first24 hours of treatment. Intrapulmonary delivery of heparin particles toachieve rapid therapeutic levels of heparin in the early stage ofthromboembolism, could also be combined with other routes ofadministration of LMWHs or heparin for prolongedantithrombotic/anticoagulant effect such as oral administration.

The HLGAG preparations can also be used to treat acute thromboembolicstroke. An acute stroke is a medical syndrome involving neurologicalinjury resulting from an ischemic event, which is an interruption orreduction in the blood supply to the brain.

An effective amount of a HLGAG preparation alone or in combination withanother therapeutic for the treatment of stroke is that amountsufficient to reduce in vivo brain injury resulting from the stroke. Areduction of brain injury is any prevention of injury to the brain whichotherwise would have occurred in a subject experiencing a thromboembolicstroke absent the treatment described herein. Several physiologicalparameters may be used to assess reduction of brain injury, includingsmaller infarct size, improved regional cerebral blood flow, anddecreased intracranial pressure, for example, as compared topretreatment patient parameters, untreated stroke patients or strokepatients treated with thrombolytic agents alone.

The pharmaceutical HLGAG preparation may be used alone or in combinationwith a therapeutic agent for treating a disease associated withcoagulation. Examples of therapeutics useful in the treatment ofdiseases associated with coagulation include anticoagulation agents,antiplatelet agents, and thrombolytic agents.

Anticoagulation agents prevent the coagulation of blood components andthus prevent clot formation. Anticoagulants include, but are not limitedto, warfarin, Coumadin, dicumarol, phenprocoumon, acenocoumarol, ethylbiscoumacetate, and indandione derivatives. “Direct thrombin inhibitors”include hirudin, hirugen, Angiomax, agatroban, PPACK, thrombin aptamers.Antiplatelet agents inhibit platelet aggregation and are often used toprevent thromboembolic stroke in patients who have experienced atransient ischemic attack or stroke. Thrombolytic agents lyse clotswhich cause the thromboembolic stroke. Thrombolytic agents have beenused in the treatment of acute venous thromboembolism and pulmonaryemboli and are well known in the art (e.g. see Hennekens et al, J AmColl Cardiol; v. 25 (7 supp), p. 18S-22S (1995); Holmes, et al, J AmColl Cardiol; v. 25 (7 suppl), p. 10S-17S(1995)).

Pulmonary embolism as used herein refers to a disorder associated withthe entrapment of a blood clot in the lumen of a pulmonary artery,causing severe respiratory dysfunction. Pulmonary emboli often originatein the veins of the lower extremities where clots form in the deep legveins and then travel to lungs via the venous circulation. Thus,pulmonary embolism often arises as a complication of deep venousthrombosis in the lower extremity veins. Symptoms of pulmonary embolisminclude acute onset of shortness of breath, chest pain (worse withbreathing), and rapid heart rate and respiratory rate. Some individualsmay experience haemoptysis.

The HLGAG preparations and methods are also useful for treating orpreventing atherosclerosis. Heparin has been shown to be beneficial inprevention of atherosclerosis in various experimental models. Due to themore direct access to the endothelium of the vascular system, inhaledheparin can be useful in prevention of atherosclerosis. Atherosclerosisis one form of arteriosclerosis that is believed to be the cause of mostcoronary artery disease, aortic aneurysm and atrial disease of the lowerextremities, as well as contributing to cerebrovascular disease.

Due to its fast absorption and variable elimination rate, HLGAG with orwithout excipients can be used as an alternative for the intravenousheparin for surgical and dialysis procedures. For example, HLGAGparticles can be inhaled prior to surgery by volunteer inhalation orpassively inhaled via trachea tube during the anesthesia prior to orduring the surgery. Surgical patients, especially those over the age of40 years have an increased risk of developing deep venous thrombosis.Thus, the use of HLGAG particles for preventing the development ofthrombosis associated with surgical procedures is contemplated. Inaddition to general surgical procedures such as percutaneousintervention (e.g., percutaneous coronary intervention (PCI)), PCTA,stents and other similar approaches, hip or knee replacement,cardiac-pulmonary by-pass surgery, coronary revascularization surgery,orthopedic surgery, and prosthesis replacement surgery, the methods arealso useful in subjects undergoing a tissue or organ transplantationprocedure or treatment for fractures such as hip fractures.

In addition, pulmonary inhalation of heparin is valuable in treatment ofrespiratory diseases such as cystic fibrosis, asthma, allergy,emphysema, adult respiratory distress syndrome (ARDS), lung reperfusioninjury, and ischemia-reperfusion injury of the lung, kidney, heart, andgut, and lung tumor growth and metastasis.

Cystic fibrosis is a chronic progressive disease affecting therespiratory system. One serious consequence of cystic fibrosis isPseudomonas aeruginosa lung infection, which by itself accounts foralmost 90% of the morbidity and mortality in cystic fibrosis.Therapeutics for treating cystic fibrosis include antimicrobials fortreating the pathogenic infection.

Heparin is also a well established inhibitor of elastase and tumorgrowth and metastasis. The aerosolized heparin particles are capable ofinhibiting elastase induced lung injury in an acute lung emphysemamodel. Asthma is a disorder of the respiratory system characterized byinflammation, narrowing of the airways and increased reactivity of theairways to inhaled agents. Asthma is frequently, although notexclusively, associated with atopic or allergic symptoms. Asthma mayalso include exercise induced asthma, bronchoconstrictive response tobronchostimulants, delayed-type hypersensitivity, auto immuneencephalomyelitis and related disorders. Allergies are generally causedby IgE antibody generation against allergens. Emphysema is a distentionof the air spaces distal to the terminal bronchiole with destruction ofalveolar septa. Emphysema arises out of elastase induced lung injury.Heparin is capable of inhibiting this elastase induced injury. Adultrespiratory distress syndrome is a term which encompasses many acutedefuse infiltrative lung lesions of diverse ideologies which areaccompanied by severe atrial hypoxemia. One of the most frequent causesof ARDS is sepsis. Inflammatory diseases include but are not limited toautoimmune diseases and atopic disorders. Other types of inflammatorydiseases which are treatable with HLGAGs are refractory ulcerativecolitis, Chrohn's disease, multiple sclerosis, autoimmune disease,non-specific ulcerative colitis and interstitial cystitis.

In one embodiment, the HLGAG preparations are used for inhibitingangiogenesis. An effective amount for inhibiting angiogenesis of theHLGAG preparation is administered to a subject in need of treatmentthereof. Angiogenesis as used herein is the inappropriate formation ofnew blood vessels. “Angiogenesis” often occurs in tumors whenendothelial cells secrete a group of growth factors that are mitogenicfor endothelium causing the elongation and proliferation of endothelialcells which results in the generation of new blood vessels. Several ofthe angiogenic mitogens are heparin binding peptides which are relatedto endothelial cell growth factors. The inhibition of angiogenesis cancause tumor regression in animal models, suggesting a use as atherapeutic anticancer agent. An effective amount for inhibitingangiogenesis is an amount of HLGAG preparation which is sufficient todiminish the number of blood vessels growing into a tumor. This amountcan be assessed in an animal model of tumors and angiogenesis, many ofwhich are known in the art. Angiogenic disorders include, but are notlimited to, neovascular disorders of the eye, osteoporosis, psoriasis,arthritis, cancer and cardiovascular disorders.

The HLGAG preparations are also useful for inhibiting neovascularizationassociated with eye disease. In another embodiment, the HLGAGpreparation is administered to treat psoriasis. Psoriasis is a commondermatologic disease caused by chronic inflammation.

HLGAG containing compositions, may also inhibit cancer cell growth andmetastasis. Thus the methods are useful for treating and/or preventingtumor cell proliferation or metastasis in a subject. The cancer may be amalignant or non-malignant cancer. Cancers or tumors include but are notlimited to biliary tract cancer; brain cancer; breast cancer; cervicalcancer; choriocarcinoma; colon cancer; endometrial cancer; esophagealcancer; gastric cancer; intraepithelial neoplasms; leukemias, lymphomas;liver cancer; lung cancer (e.g. small cell and non-small cell);melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreaticcancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas.

A subject in need of cancer treatment may be a subject who has a highprobability of developing cancer. These subjects include, for instance,subjects having a genetic abnormality, the presence of which has beendemonstrated to have a correlative relation to a higher likelihood ofdeveloping a cancer and subjects exposed to cancer-causing agents suchas tobacco, asbestos, or other chemical toxins, or a subject who haspreviously been treated for cancer and is in apparent remission.

Other Embodiments

This invention is further illustrated by the following examples thatshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example 1 Cloning and Recombinant Expression of B.thetaiotaomicron GAG Lyase I

The B. thetaiotaomicron GAG lyase I sequence (FIG. 1; SEQ ID NO:1),which is approximately 1251 nucleotides long contains a predictedmethionine-initiated coding sequence of about 1179 nucleotides,including the termination codon (SEQ ID NO:1 in FIG. 1A). The codingsequence encodes a 392 amino acid protein (SEQ ID NO:2 in FIG. 1B).

The B. thetaiotaomicron GAG lyase amino acid sequence shares somestructural homology to the F. heparinum heparinase I sequence. Acomparison of the amino acid sequences of the two lyases is shown inFIG. 2.

The B. thetaiotaomicron GAG lyase gene was cloned by PCR using genomicDNA from B. thetaiotaomicron obtained from the American Type CultureCollection (ATCC), catalog no. 29148D. DNA oligonucleotide primers forM17 variant were synthesized by Integrated DNA technologies, Inc. (IDT)according to the following nucleotide sequences: 1) 5′CATATGCTGACTGCTCAGACTAAAAATAC 3′ (forward primer) (SEQ ID NO:11); 2) 5′CTCGAGTTATCTTTCCGAATATCCTGCGAGAT 3′ (reverse primer) (SEQ ID NO:12).Primers were designed to introduce NdeI and XhoI endonucleaserestriction sites at the 5′ and 3′ ends, respectively. The resultinggene sequence was cloned into pET28a bacterial expression plasmid (EMDBiosciences) as an NdeI-XhoI fragment for subsequent recombinantexpression into E. coli strain BL21 (DE3), as an engineered fusionprotein containing the sequence MGSSHHHHHHSSGLVPRGSH (SEQ ID NO:13)fused to the amino terminus of the B. thetaiotaomicron GAG lyasebeginning at the methionine at position 17 (M17).

A B. thetaiotaomicron GAG lyase variant with a modified amino terminusthat begins at position glutamine 26 (Q26) of the protein sequencelisted in SEQ ID NO:2, was cloned into pET28a for recombinant expressionas a fusion protein. The amino acid sequence and nucleic acid sequenceencoding the Q26 variant are provided in SEQ ID NOs: 4 and 3,respectively DNA oligonucleotide primers for Q26 variant weresynthesized by Integrated DNA technologies, Inc. (IDT) according to thefollowing nucleotide sequences: 1) 5′ CAT ATG CAA ACA CTG ATG CCA CTCACC GAA 3′ (forward primer) (SEQ ID NO:41) and 5′CTCGAGTTATCTTTCCGAATATCCTGCGAGAT 3′ (reverse primer) (SEQ ID NO:12).

Both the full length, M17, and Q26 B. thetaiotaomicron GAG lyase fusionproteins were recombinantly expressed in E. coli, yielding soluble,highly active enzyme that was fully capable of cleaving heparin andheparan sulfate (see Example 2 below). [Sequence verified plasmid pET28containing either the M17 coding sequence or Q26 coding sequence wastransformed into BL21 (DE3). 2 liter cultures were grown at roomtemperature (˜22-25° C.) in LB media supplemented with 40 μg/mLkanamycin. Protein expression was induced with 500 μM IPTG added at anA₆₀₀ of 1.0. Induced cultures were allowed to grow for 15-18 hours atroom temperature.

Recombinant B. thetaiotaomicron GAG lyase purification. Bacterial cellswere harvested by centrifugation at 6000×g for 15 minutes andresuspended in 30 mL of binding buffer (50 mM Na₂HPO₄, pH 7.9, 0.5 MNaCl, and 5 mM imidazole). Lysis was initiated by the addition of 0.1mg/mL lysozyme (20 minutes at room temperature) followed by intermittentsonication in an ice-water bath using a Misonex XL sonicator at 40-50%output. The crude lysate was fractionated by low-speed centrifugation(20,000×g; 4° C.; 30 minutes) and the supernatant was filtered through a0.45 micron filter. The 6×-His recombinant B. thetaiotaomicron GAG lyasewas purified by Ni⁺² chelation chromatography on a 5 mL Hi-Trap column(GE Healthcare) pre-charged with 200 mM NiSO₄ and subsequentlyequilibrated with binding buffer. The column was run at a flow rate ofapproximately 3 ml/minute that included an intermediate wash step with50 mM imidazole. The lyase enzyme was eluted from the column in 5 mLfractions using high imidazole elution buffer (50 mM Na₂HPO₄, pH 7.9,0.5 M NaCl, and 250 mM imidazole). These enzymes can also be purifiedusing purification tags such as GST, MBP, Trx, DsbC, NusA or biotin

The resulting peak was buffer exchanged on a Sephadex G-25 columnequilibrated with 20 mM Na₂HPO₄, pH 6.8, 150 mM NaCl and subsequentlysubjected to cation exchange chromatography using a source 15S resin (GEhealthcare) and applying a linear salt gradient from 0.05 M-1 M NaCl.

Protein concentrations were determined by the Bio-Rad protein assay andconfirmed by UV spectroscopy. Protein purity was assessed by SDS-PAGEfollowed by Coomassie Brilliant Blue staining and/or Sypro Ruby Red(Invitrogen).

Example 2 Distinct Heparan Sulfate Substrate Specificities of B.thetaiotaomicron GAG Lyase I and F. heparinum Heparinases I and II

The cleavage patterns and thereby the substrate specificities ofrecombinant B. thetaiotaomicron GAG lyase I and F. heparinum heparinasesI and II were compared using heparan sulfate as a substrate. 200 μg of“HI” fraction of heparan sulfate (Celsus Labs) from porcine intestinalmucosa was digested with recombinant B. thetaiotaomicron GAG lyase Iunder conditions favorable to ensure a complete digestion. The HS wascontacted with about 50 μg B-thetaiotaomicron GAG lyase I, 50 mM sodiumphosphate, 100 mM NaCl, pH 8.0 at 37° C. for 18 hours. The lyasedigestion products were analyzed by HPLC using strong anionchromatography (SAX-HPLC). SAX-HPLC conditions were as follows: 50 μgsamples was injected at 1 mg/ml into a 4×250 mm CarboPac PA1 analyticalscale column (Dionex Corporation). The flow rate was 1 ml/min. Themobile phase was 0.2M to 2 M NaCl in water, pH 3.5, gradient over 120minutes. The column was preequilibrated with 0.2 M NaCl for 10 minutes.The results were compared with the results of the same experiment exceptthat F. heparinum heparinase I was used to digest the heparan sulfate.Briefly, The HS was contacted with about 50 μg F. heparinum heparinaseI, 25 mM sodium acetate, 1 mM calcium acetate, 5% glycine, pH 7.0 at 30°C. for 18 hours. The digestion profile for heparinase I is very similarto the profile for B. thetaiotaomicron GAG lyase I, except that novelpeaks are present in the B. thetaiotaomicron GAG lyase I profile thatare not present in the heparinase I profile, demonstrating that thelyases have non-identical substrate specificities. Further, the traceprofile using B. thetaiotaomicron GAG lyase I was compared to theresults of the same experiment except that F. heparinum heparinase IIwas used to digest the heparan sulfate. Briefly, The HI was contactedwith about 50 μg F. heparinum heparinase II, 25 mM sodium acetate, 1 mMcalcium acetate, pH 7.0 at 37° C. for 18 hours. In this case, thedigestion profile using heparinase II is very much distinct from thedigestion profile of B. thetaiotaomicron GAG lyase and F. heparinumheparinase I. These data demonstrate that the B. thetaiotaomicron GAGlyase substrate specificity is distinct from the specificities of F.heparinum heparinases I and II, but is more “heparin like” (e.g., moresimilar to F. heparinum heparinase I) than “heparan sulfate-like” (e.g.,it is less like F. heparinum heparinase II).

Example 3 Depolymerization and Neutralization of ARIXTRA® by B.thetaiotaomicron GAG Lyase I

Recombinant B. thetaiotaomicron GAG lyase I can cleave and therebyneutralize the ATIII pentasaccharide ARIXTRA® into a pentasulfatedtrisaccharide and an unsaturated disulfated disaccharide. ARIXTRA® is ananti-thrombotic drug that acts as a selective inhibitor of Factor Xa, acomponent of the coagulation cascade. Depolymerization of ARIXTRA® isunequivocally demonstrated by matrix assisted laser desorptionionization mass spectrometry (MALDI-MS) (FIG. 7). Panel A shows the scanof ARIXTRA® in the absence of a lyase. The structure of ARIXTRA® is alsoshown. Panel B shows the scan after cleavage of ARIXTRA® with B.thetaiotaomicron GAG lyase. Briefly, 1 mg/ml ARIXTRA® in a 20 μLreaction volume was treated with 5 μg B. thetaiotaomicron GAG lyase 1.25mM sodium acetate, 1 mM calcium acetate, pH 7.0 at 37° C. for 2 hours.Note the disappearance in panel B of mass 4723.7 Da (net mass=1506 Da)present in panel A with concomitant appearance of mass 4133.2 Da (netmass=915.6 Da). The latter mass represents the pentasulfatedtrisaccharide cleavage product. In cleaving Arixtra into two smallerfragments, the drug's anti-Xa activity is effectively neutralized by theB. thetaiotaomicron GAG lyase.

Example 4 Cloning and Recombinant Expression of B. thetaiotaomicron GAGLyase II

The complete coding sequence of a B. thetaiotaomicron GAG lyase II(herein described as “full-length gene”) as well as the two variantsdescribed herein were cloned by PCR using genomic DNA from Bacteroidesthetaiotaomicron as obtained from American Type Culture Collection(ATCC), catalog no. 29148D. DNA oligonucleotide primers were synthesizedby Integrated DNA technologies (IDT), Inc. according to the followingnucleotide sequences: 1) For the full-length gene: 5′ CAT ATG AAT AAAACC CTG AAA TAT ATC GTC CTG 3′ (forward primer) (SEQ ID NO:14), 5′ CTCGAG TTA TAA TTT ATA TTT TAA TGA CTG TTT CTT GC 3′ (reverse primer) (SEQID NO:15); 2) Gene encoding variant No. 1 (amino terminal truncation toremove putative signal sequence): 5′ CAT ATG CAA GAG TTG AAA AGC GAG GTATTC TCG 3′ (forward primer) (SEQ ID NO:16), 5′ CTC GAG TTA TAA TTT ATATTT TAA TGA CTG TTT CTT GC 3′ (note: same reverse primer listed above asfor full-length gene) (SEQ ID NO:15). Primers were designed to introduceNde 1 and Xho 1 endonuclease restriction sites at the 5′ and 3′ ends,respectively. Cloning of described gene sequence into pET28b bacterialexpression plasmid (EMD Biosciences) as an Nde 1-Xho 1 fragment forsubsequent recombinant expression into E. coli strain BL21 (DE3) asengineered fusion protein containing the sequenceMGSSHHHHHHSSGLVPRGSHMNKTLKY . . . KVNGKKQSLKYKL (SEQ ID NO:17) orMGSSHHHHHHSSGLVPRGSHMQELKSEVF . . . KVNGKKQSLKYKL (SEQ ID NO:18) for thefull-length gene and variant 1 (the Q23 variant, SEQ ID NO:8),respectively (B. thetaiotaomicron GAG lyase sequence is denoted inbold). See FIG. 4 for complete sequence.

Another variant, the K169 variant (SEQ ID NO:10) represents anengineered deletion of 18 contiguous amino acids comprising an internalregion within the protein and possessing the following linear sequence:KMDKKEYELVSDGKIKGE. (SEQ ID NO:19) Deletion of this region in the genesequence (FIG. 5A) and in the corresponding protein sequence (FIG. 5B)is noted by grey shading. Deletion of this region at the DNA level wasaccomplished by PCR-based mutagenesis using the Quick-change kit(Stratagene) in accordance with the manufacturer's instructions.Mutagenesis primers used to make this deletion at the gene (DNA) levelwere of the following sequence: 5′ GG ATT AAA AAG AAT CCG TTG GTG GAAAAT GTA CGT TTC GC 3′ (SEQ ID NO:20) and 5′ CC TAA TTT TTC TTA GGC AACCAC CTT TTA CAT GCA AAG CG 3′ (SEQ ID NO:21) corresponding to the senseand anti-sense strands, respectively. Recombinant expression of thisdescribed gene variant in E. coli likewise based on the pET-basedexpression for recombinant expression was also achieved. Purificationwas carried out largely as described for the B. thetaiotaomicron GAGlyase I.

Preliminary biochemical characterization of this variant indicates thatdeletion of described amino acids is not deleterious to the solubleexpression of the enzyme nor to its ability to cleave both heparin andheparan sulfate. It does suggest, however a potential difference in thecatalytic efficiency and/or substrate specificity of this enzyme variantrelative to the full-length protein.

Example 5 Cloning, Recombinant Expression and Purification of B.thetaiotaomicron GAG Lyase III

GAG lyase III gene was cloned by PCR using genomic DNA from Bacteroidesthetaiotaomicron obtained from American Type Culture Collection (ATCC),catalog number 29148D. The nucleotide sequence (SEQ ID NO: 28) of afull-length gene of at least 2622 base pairs is shown in FIG. 8. Theamino acid sequence (SEQ ID NO: 29) encoding a polypeptide of at least873 amino acids existing in the linear sequence is shown in FIG. 9.

DNA oligonucleotide primers were synthesized by Integrated DNAtechnologies (IDT), Inc. according to the following nucleotidesequences: 1) 5′CATATGATGAAACAACGATATTATATTTTC 3′ (forward primer) (SEQID NO: 30); 2) 5′GGATCCTCGAGTTATATCTCAAAATCCGGTAAATAGTC 3′ (reverseprimer) (SEQ ID NO: 31). Primers were designed to introduce Nde 1 andBam H1/Xho 1 endonuclease restriction sites at the 5′ and 3′ ends,respectively. The described gene sequence (SEQ ID NO:28) was cloned intopET28a bacterial expression plasmid (EMD Biosciences) as an Nde 1-Xho 1fragment for subsequent recombinant expression in E. coli strain BL21(DE3), engineered as a 6× Histidine fusion protein.

Likewise, a gene variant of GAG lyase III (SEQ ID NO:33 and 34) withmodified amino terminus that begins at position glutamine 23 (Q23) inthe protein sequence listed (SEQ ID NO:29) was cloned into pET28a forrecombinant expression. To do so, the following forward (5′) primer wasused: CATATGCAGAAAAGCATCCTGCGTCTGAGT 3′ (SEQ ID NO:35).

The primary amino acid sequence of the cloned enzyme was compareddirectly with four functionally-related lyases from both Bacteroidesthetaiotaomicron and Flavobacterium heparinum. The multiple sequencealignment (made using CLUSTALW program) is depicted in FIG. 10. Thealignment shows that the GAG lyase III disclosed herein is distinct fromthe other enzymes to which it is being compared.

The enzyme was recombinantly expressed in E. coli as a highly soluble,active amino-terminal variant beginning at Q23 (SEQ ID NO: 33 and 34),as shown in FIG. 9. The Q23 variant was readily expressed in E. coli asa highly soluble enzyme. Expression of this enzyme as an amino terminal6× histidine fusion protein also facilitated purification in essentiallya single purification step. Additional purification included cationexchange chromatography on a Source 15S column utilizing a lineargradient from 0.05 M to 1 M NaCl. Other purification methods that can beused to obtain these enzymes include affinity purification tags such asGST, MBP, Trx, DsbC, NusA or biotin.

Example 6 Analysis of Function and Enzyme Activity of B.thetaiotaomicron GAG Lyase III

The ability of the recombinant GAG lyase III to cleave heparin-likeglycosaminoglycans was evaluated. Heparinase II and heparinase III fromFlavobacterium heparinum were included for comparison. Cleavage of four“heparin-like” substrates: porcine intestinal heparin, two differentheparan sulfates (designated HI and HO, each with a varying degree ofsulfation), and enoxaparin (a low-molecular weight pharmaceuticalheparin), was analyzed. Biochemical enzyme activity was assessed bothfor extent of cleavage (product formation) as measured by UV absorbanceat 232 nm, as well as for specificity. The latter parameter was assessedby fractionation of the di-, tetra-, and higher oligosaccharide productsby capillary electrophoresis (CE).

Total enzyme activity of the B. thetaiotaomicron GAG lyase III issummarized in FIG. 11 These data show that GAG lyase III exhibited apreference for highly sulfated, “heparin-like” GAGs and their lowermolecular weight derivatives. At the same time, this enzyme was able tocleave GAGs with lesser sulfation, i.e., GAGs that are more “heparansulfate-like.” At the same time, the substrate profile depicted in FIG.11 demonstrates that the specificity of that lyase was distinct fromboth heparinase II and heparinase III derived from F. heparinum.

The distinction between B. thetaiotaomicron GAG lyase III and heparinaseII from F. heparinum was analyzed further and is shown in FIG. 12.Cleavage of three substrates: heparin, and two different heparansulfates (designated HI and HO, each with a varying degree of sulfation)was analyzed. The cleavage products were fractionated by capillaryelectrophoresis and monitored by absorbance at 232 nm (Y axis). Thesedata indicates that while the two enzymes are functionally related,their substrate specificities are not identical.

Example 7 Cloning, Recombinant Expression and Purification of B.thetaiotaomicron GAG Lyase IV

The B. thetaiotaomicron GAG lyase IV sequence (FIG. 13, SEQ ID NO:36),which is approximately 2109 nucleotides long encodes a polypeptide of atleast 702 amino acids (FIG. 14, SEQ ID NO:37).

The GAG lyase IV gene was cloned by PCR using genomic DNA from B.thetaiotaomicron as obtained from American Type Culture Collection(ATCC), catalog no. 29148). DNA oligonucleotide primers were synthesizedby Integrated DNA technologies (IDT), Inc. according to the followingnucleotide sequences:

1) 5′CCA TGG CAT ATG AAG AAC ATC TTC TTT ATT TGC 3′ (forward primer, SEQID NO:32);

2) 5′CTC GAG TTA TAA GGT ATA AGA TAA TGT ATG TGT 3′ (reverse primer, SEQID NO:40).

Primers were designed to introduce Nde 1 and Xho 1 endonucleaserestriction sites as indicated in bold. Three amplified gene wassubcloned into the T7-based expression vector pET28 for heterologousexpression in E. coli as an engineered fusion in which a 6× histidinetag is present at the amino terminus as a means to purify therecombinantly expressed protein. Other methods can be used to purifythis enzyme including the use of GST, MBP, Trx, DsbC, NusA andbiotinylation. A variant of this gene was also constructed, namely onein which the first 19 amino acids representing a putative bacterialsecretion signal was removed. This variant (known as D20) begins ataspartate 20 (asp 20).

The primary amino acid sequence of the cloned enzyme was compareddirectly with four functionally related lyases from both B.thetaiotaomicron and Flavobacterium heparinum. The multiple sequencealignment is depicted in FIG. 15. The amino acid sequence correspondingto B. thetaiotaomicron lyase IV exhibits approximately 30% identity withheparin lyase III from F. heparinum. Thus, from this alignment, it isclear that the GAG lyase described in this disclosure is distinct fromthe other enzymes to which it is being compared.

The functionality of this putative lyase was also examined. Inparticular, the ability of the recombinant B. thetaiotaomicron lyase IVenzyme to cleave heparin-like glycosaminoglycans of differingcomposition, particularly as it relates to sulfation density wasevaluated. In this experiment, three GAG substrates were screened:heparin from pig intestinal mucosa (PM), “HI” fraction of heparansulfate likewise isolated from pig intestinal mucosa (HI-HS), andso-called “HO” fraction of heparan sulfate representing the lowestsulfation density. Biochemical enzyme activity was assessed both forrate of cleavage as measured in a real-time, UV-based kinetic assay inaddition to the extent of cleavage (product formation) as measured by UVabsorbance at 232 nm. Data are summarized in FIG. 18 and representrelative values normalized to the highest activity reported. Based onthese data, this recombinantly expressed GAG lyase exhibits a preferencefor GAGs of medium sulfation density. This preference may be broadlydescribed as heparan sulfate-like rather than heparin-like.

The B. thetaiotaomicron GAG lyase IV may also have a substratespecificity that is unique when compared to other heparan sulfate lyasessuch as heparinase III from F. heparinum—or even other “heparinaseIII-like” enzymes from Bacteroides. In particular, this enzyme maycleave disaccharides not commonly found in either heparin or mostfractions of heparan sulfate, for example, I_(2S)H_(NAc). It is alsopossible that this enzyme may cleave heparin in such a manner as topreserve, at least in part, the AT-III binding site.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of specifically cleaving a heparin or a heparan sulfate,comprising: selecting an isolated B. thetaiotaomicron HSGAG lyasepolypeptide I, or a functional fragment thereof, that cleaves aglycosidic linkage between a glucosamine having a sulfate at position 3and a sulfated uronic acid of the heparin or heparan sulfate, whereinthe B. thetaiotaomicron HSGAG lyase I polypeptide comprises a) an aminoacid sequence which is at least 90% identical to the amino acid sequenceof SEQ ID NO:2; b) the amino acid sequence of SEQ ID NO:2, 4 or 23; andc) an amino acid sequence which differs by at least 1 amino acid but notmore than 15 amino acids from the amino acid sequence of SEQ ID NO:2,and contacting the heparin or the heparan sulfate with the B.thetaiotaomicron HSGAG lyase polypeptide I, to thereby cleave theheparin or heparan sulfate.
 2. The method of claim 1, further comprisingcontacting the heparin or heparan sulfate with an isolated B.thetaiotaomicron HSGAG lyase II polypeptide, or functional fragmentthereof.
 3. The method of claim 1, wherein the B. thetaiotaomicron HSGAGlyase I polypeptide is encoded by a nucleotide sequence selected fromthe group consisting of: a) a nucleic acid molecule comprising afragment of at least 800 nucleotides of the nucleotide sequence of SEQID NO: 1, 3 or 22; b) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:2, 4 or 23;and c) a nucleic acid molecule which encodes a fragment of a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, 4 or 23, wherein thefragment comprises at least 300 contiguous amino acids of SEQ ID NO:2, 4or
 23. 4. The method of claim 1 or 3, wherein the B. thetaiotaomicronHSGAG lyase I cleaves a heparin at one or more glycosidic linkages of2-O sulfated uronic acids.
 5. The method of claim 1, wherein the heparinor heparan sulfate is cleaved into di-, tri-, tetra-, penta-, hexa-,octa-, deca-saccharides, and combinations thereof.
 6. The method ofclaim 1, further comprising contacting the heparin or heparan sulfatewith one or more HLGAG degrading enzyme other than the B.thetaiotaomicron HSGAG lyase I polypeptide.
 7. The method of claim 6,wherein the HLGAG degrading enzyme is selected from Flavobacteriumheparinum heparinase I, Flavobacterium heparinum heparinase II,Flavobacterium heparinum heparinase III, Flavobacterium heparinumheparinase IV, heparanase, sulfatase, delta 4,5 glucuronidase andfunctional fragments thereof.
 8. The method of claim 1, furthercomprising determining if a structural signature is absent or present inthe cleaved heparin or heparin sulfate.
 9. The method of claim 8,further comprising comparing the absence or presence of the structuralsignature or the amount of the structural signature of the heparin orheparin sulfate to a preselected value.
 10. The method of claim 9,wherein the preselected value is the absence, presence or amount of thestructural signature present in a commercially available heparin orheparin sulfate.
 11. The method of claim 10, wherein the commerciallyavailable heparin is selected from: unfractionated heparin, enoxaparin,dalteparin, certoparin, ardeparin, nadroparin, parnaparin, reviparin,tinzaparin, and Fondaparinux.
 12. The method of claim 9, furthercomprising selecting or discarding the heparin or heparin sulfate as aresult of the determination.
 13. The method of claim 8, wherein thestructural signature is determined by one or more of mass spectroscopy,NMR spectroscopy, gel electrophoresis, capillary electrophoresis,reverse-phase column chromatography, and ion-pair HPLC.
 14. The methodof claim 10, wherein the structural signature is one or more of thefollowing structures:


15. A method of producing an enoxaparin preparation, the methodcomprising the steps of: comprising: selecting an isolated B.thetaiotaomicron HSGAG lyase polypeptide I, or a functional fragmentthereof, that cleaves a glycosidic linkage between a glucosamine havinga sulfate at position 3 and a sulfated uronic acid of heparin or heparansulfate, wherein the B. thetaiotaomicron HSGAG lyase I polypeptidecomprises a) an amino acid sequence which is at least 90% identical tothe amino acid sequence of SEQ ID NO:2; b) the amino acid sequence ofSEQ ID NO:2, 4 or 23; and c) an amino acid sequence which differs by atleast 1 amino acid but not more than 15 amino acids from the amino acidsequence of SEQ ID NO:2, and contacting the enoxaparin preparation withthe B. thetaiotaomicron HSGAG lyase polypeptide I, to thereby cleave theenoxaparin preparation; determining if a structural signature is absentor present in the cleaved enoxaparin preparation; and based upon thedetermination, further processing the enoxaparin batch by a methodincluding selecting or discarding the enoxaparin preparation.